Antibodies, variable domains &amp; chains tailored for human use

ABSTRACT

The invention relates to the provision of antibody therapeutics and prophylactics that are tailored specifically for human use. The present invention provides libraries, vertebrates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. Furthermore, the invention relates to methods of using the vertebrates to isolate antibodies or nucleotide sequences encoding antibodies. Antibodies, heavy chains, polypeptides, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

CROSS REFERENCE

This is a Continuation of U.S. Ser. No. 14/052,259 filed Oct. 11, 2013,which is a Continuation of PCT/GB2012/052296 filed Sep. 18, 2012, whichclaims priority to GB 1116122.1 filed Sep. 19, 2011, GB 1116120.5 filedSep. 19, 2011, GB 1203257.9 filed Feb. 24, 2012, GB 1204592.8 filed Mar.15, 2012, GB 1205702.2 filed Mar. 29, 2012, GB 1208749.0 filed May 18,2012 and GB 1211692.7 filed Jul. 2, 2012, all of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the provision of antibody therapeuticsand prophylactics that are tailored specifically for human use.

The present invention provides libraries, vertebrates and cells, such astransgenic mice or rats or transgenic mouse or rat cells. Furthermore,the invention relates to methods of using the vertebrates to isolateantibodies or nucleotide sequences encoding antibodies. Antibodies,heavy chains, polypeptides, nucleotide sequences, pharmaceuticalcompositions and uses are also provided by the invention.

BACKGROUND

The state of the art provides non-human vertebrates (eg, mice and rats)and cells comprising transgenic immunoglobulin loci, such locicomprising human variable (V), diversity (D) and/or joining (J)segments, and optionally human constant regions. Alternatively,endogenous constant regions of the host vertebrate (eg, mouse or ratconstant regions) are provided in the transgenic loci. Methods ofconstructing such transgenic vertebrates and use of these to generateantibodies and nucleic acids thereof following antigen immunisation areknown in the art, eg, see U.S. Pat. No. 7,501,552 (Medarex), U.S. Pat.No. 5,939,598 (Abgenix), U.S. Pat. No. 6,130,364 (Abgenix), WO02/066630(Regeneron), WO2011004192 (Genome Research Limited), WO2009076464,WO2009143472 and WO2010039900 (Ablexis), the disclosures of which areexplicitly incorporated herein. Such transgenic loci in the art includevarying amounts of the human V(D) J repertoire. Existing transgenicimmunoglobulin loci are based on a single human DNA source. Thepotential diversity of human antibody variable regions in non-humanvertebrates bearing such transgenic loci is thus confined.

The inventors considered that it would be desirable to tailor thegenomes of these transgenic non-human vertebrates (and thus antibody andantibody chain products of these) to address the variability—andcommonality—in the natural antibody gene usage of humans. The inventorswanted to do this in order to better address human use of antibody-basedtherapeutic and prophylactic drugs.

It would be desirable also to provide for novel and potentially expandedrepertoire and diversity of human variable regions in transgenicimmunoglobulin loci and non-human vertebrates harbouring these, as wellas in antibodies produced following immunisation of such animals.

SUMMARY OF THE INVENTION

The present invention has been developed from extensive bioinformaticsanalysis of natural antibody gene segment distributions across a myriadof different human populations and across more than two thousand samplesfrom human individuals. The inventors have undertaken this huge task tomore thoroughly understand and design non-human vertebrate systems andresultant antibodies to better address human medical therapeutics as awhole, as well as to enable rational design to address specific ethnicpopulations of humans. Using such rational design, the inventors haveconstructed transgenic non-human vertebrates and isolated antibodies,antibody chains and cells expressing these in a way that yields productsthat utilise gene segments that have been purposely included on thebasis of the human bioinformatics analysis. The examples illustrateworked experiments where the inventors isolated many cells andantibodies to this effect.

The invention also relates to synthetically-extended &ethnically-diverse superhuman immunoglobulin gene repertoires. Thepresent invention thus provides for novel and potentially expandedsynthetic immunoglobulin diversities, thus providing a pool of diversityfrom which human antibody therapeutic leads can be selected. Thisexpanded pool is useful when seeking to find antibodies with desirablecharacteristics, such as relatively high affinity to target antigenwithout the need for further affinity maturation (eg, using laborious invitro techniques such as phage or ribosome display), or improvedbiophysical characteristics, or to address targets and new epitopes thathave previously been difficult to address with antibodies are notreached by prior antibody binding sites.

The invention also provides for diversity that is potentially biasedtowards variable gene usage common to members of a specific humanpopulation, which is useful for generating antibodies for treatingand/or preventing diseases or conditions within such population. Thisability to bias the antibody repertoire allows one to tailor antibodytherapeutics with the aim of more effectively treating and/or preventingdisease or medical conditions in specific human populations.

The present inventors realised the possibility of providingimmunoglobulin gene segments from disparate sources in transgenic loci,in order to provide for novel and potentially-expanded antibodydiversities from which antibody therapeutics (and antibody toolreagents) could be generated. This- opens up the potential of transgenichuman-mouse/rat technologies to the possibility of interrogatingdifferent and possibly larger antibody sequence-spaces than has hithertobeen possible.

In rationally designing transgenic antibody loci, as well as antibodiesand antibody chains, the inventors also realised that a relatively longHCDR3 length (at least 20 amino acids) is often desirable to addressepitopes. For example, naturally-occurring antibodies have been isolatedfrom humans infected with infectious disease pathogens, such antibodieshaving a long HCDR3 length. Neutralising antibodies have been found inthis respect. A long HCDR3 length would be desirable to address otherantigens (eg, receptor clefts or enzyme active sites), not just limitedto infectious disease pathogens, and thus the inventors realised thegeneral desirability of the possibility of engineering transgenic locito be able to produce long HCDR3 antibodies and heavy chains. Theinventors, through laborious execution of bioinformatics on in excess of2000 human DNA samples via the 1000 Genomes project together withrational sequence choices, identified that the inclusion of the specifichuman gene segment variant JH6*02 is desirable for producing long HCDR3antibodies and chains.

Additional rational design and bioinformatics has led the inventors torealise that specific human constant region variants are conservedacross many diverse human populations. The inventors realised that thisopens up the possibility of making a choice to humanise antibodies,chains and variable domains by using such specific constant regions inproducts, rather than arbitrarily choosing the human constant region (ora synthetic version of a human constant region). This aspect of theinvention also enables one to tailor antibody-based drugs to specifichuman ethnic populations, thereby more closely matching drug to patient(and thus disease setting) than has hitherto been performed. It can be aproblem in the state of the art that antibodies are humanised with anarbitrary choice of human constant region (presumably derived from one(often unknown) ethnic population or non-naturally occurring) that doesnot function as well in patients of a different human ethnic population.This is important, since the constant region has the major role inproviding antibody effector functions, eg, for antibody recycling,cellular and complement recruitment and for cell killing.

To this end, in a first configuration of the invention, there isprovided

First Configuration

A non-human vertebrate or vertebrate cell (optionally an ES cell orantibody-producing cell) comprising a genome having a superhumanimmunoglobulin heavy chain human VH and/or D and/or J gene repertoire.

A non-human vertebrate or vertebrate cell (optionally an ES cell orantibody-producing cell) comprising a genome having a superhumanimmunoglobulin light chain human VL gene repertoire; optionally whereinthe vertebrate or cell is according to the first configuration.

A non-human vertebrate or vertebrate cell (optionally an ES cell orantibody-producing cell) whose genome comprises a transgenicimmunoglobulin locus (eg, a heavy chain locus or a light chain locus),said locus comprising immunoglobulin gene segments according to thefirst and second human immunoglobulin gene segments (optionally Vsegments) as mentioned below operably connected upstream of animmunoglobulin constant region; optionally wherein the genome ishomozygous for said transgenic immunoglobulin locus;

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional V gene segments; and/or

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional D gene segments; and/or

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional J gene segments.

A transgenic non-human vertebrate (eg, a mouse or rat) or vertebratecell (optionally an ES cell or antibody-producing cell) whose genomecomprises a transgenic immunoglobulin locus comprising a plurality ofhuman immunoglobulin gene segments operably connected upstream of anon-human vertebrate constant region for the production of a repertoireof chimaeric antibodies, or chimaeric light or heavy chains, having anon-human vertebrate constant region and a human variable region;wherein the transgenic locus comprises one or more human immunoglobulinV gene segments, one or more human J gene segments and optionally one ormore human D gene segments, a first (optionally a V segment) of saidgene segments and a second (optionally a V segment) of said genesegments being different and derived from the genomes of first andsecond human individuals respectively, wherein the individuals aredifferent; and optionally not related; optionally wherein theimmunoglobulin locus comprises more than the natural human complement offunctional V gene segments; and/or

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional D gene segments; and/or

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional J gene segments.

A transgenic non-human vertebrate (eg, a mouse or rat) or vertebratecell (optionally an ES cell or antibody-producing cell) whose genomecomprises first and second transgenic immunoglobulin loci, each locuscomprising a plurality of human immunoglobulin gene segments operablyconnected upstream of a non-human vertebrate constant region for theproduction of a repertoire of chimaeric antibodies, or chimaeric lightor heavy chains, having a non-human vertebrate constant region and ahuman variable region;

wherein (i) the first transgenic locus comprises one or more humanimmunoglobulin V gene segments, one or more human J gene segments andoptionally one or more human D gene segments, (ii) the second transgeniclocus comprises one or more human immunoglobulin V gene segments, one ormore human J gene segments and optionally one or more human D genesegments; and (iii) wherein a first (optionally a V) gene segment ofsaid first locus and a second (optionally a V) gene segment of saidsecond gene locus are different and derived from the genomes of firstand second human individuals respectively, wherein the individuals aredifferent; and optionally not related;

optionally wherein the first and second loci are on differentchromosomes (optionally chromosomes with the same chromosome number) insaid genome;

optionally wherein each immunoglobulin locus comprises more than thenatural human complement of functional V gene segments; and/or

optionally wherein each immunoglobulin locus comprises more than thenatural human complement of functional D gene segments; and/or

optionally wherein each immunoglobulin locus comprises more than thenatural human complement of functional J gene segments.

A method of constructing a cell (eg, an ES cell) according to theinvention, the method comprising

(a) identifying functional V and J (and optionally D) gene segments ofthe genome sequence of a (or said) first human individual;

(b) identifying one or more functional V and/or D and/or J gene segmentsof the genome sequence of a (or said) second human individual, whereinthese additional gene segments are not found in the genome sequence ofthe first individual;

(c) and constructing a transgenic immunoglobulin locus in the cell,wherein the gene segments of (a) and (b) are provided in the locusoperably connected upstream of a constant region.

In one embodiment, the gene segment(s) in step (b) are identified froman immunoglobulin gene database selected from the 1000 Genomes, Ensembl,Genbank and IMGT databases.

Throughout this text, Genbank is a reference to Genbank release number185.0 or 191.0; the 1000 Genomes database is Phase 1, release v3, 16Mar. 2012; the Ensembl database is assembly GRCh37.p8 (10/04/2012); theIMGT database is available at www.imgt.org.

In one embodiment, the first and second human individuals are members offirst and second ethnic populations respectively, wherein thepopulations are different, optionally wherein the human immunoglobulingene segment derived from the genome sequence of the second individualis low-frequency (optionally rare) within the second ethnic population.

This configuration of the invention also provides a method of making atransgenic non-human vertebrate (eg, a mouse or rat), the methodcomprising

(a) constructing an ES cell (eg, a mouse C57BL/6N, C57BL/6J, 129S5 or129Sv strain ES cell) by carrying out the method above;

(b) injecting the ES cell into a donor non-human vertebrate blastocyst(eg, a mouse C57BL/6N, C57BL/6J, 129S5 or 129Sv strain blastocyst);

(c) implanting the blastocyst into a foster non-human vertebrate mother(eg, a C57BL/6N, C57BL/6J, 129S5 or 129Sv strain mouse); and

(d) obtaining a child from said mother, wherein the child genomecomprises a transgenic immunoglobulin locus.

In one embodiment, the invention provides a method of isolating anantibody that binds a predetermined antigen (eg, a bacterial or viralpathogen antigen), the method comprising immunising a non-humanvertebrate according to the invention.

Second Configuration

A library of antibody-producing transgenic cells whose genomescollectively encode a repertoire of antibodies, wherein

(a) a first transgenic cell expresses a first antibody having a chainencoded by a first immunoglobulin gene, the gene comprising a firstvariable domain nucleotide sequence produced following recombination ofa first human unrearranged immunoglobulin gene segment;

(b) a second transgenic cell expresses a second antibody having a chainencoded by a second immunoglobulin gene, the second gene comprising asecond variable domain nucleotide sequence produced followingrecombination of a second human unrearranged immunoglobulin genesegment, the first and second antibodies being non-identical;

(c) the first and second gene segments are different and derived fromthe genome sequences of first and second human individuals respectively,wherein the individuals are different; and optionally not related;

(d) wherein the cells are non-human vertebrate (eg, mouse or rat) cells.

In one embodiment, the first and second human individuals are members offirst and second ethnic populations respectively, wherein thepopulations are different; optionally wherein the ethnic populations areselected from those identified in the 1000 Genomes database.

In another embodiment, the second human immunoglobulin gene segment is apolymorphic variant of the first human immunoglobulin gene segment;optionally wherein the second gene segment is selected from the groupconsisting of a gene segment in any of Tables 1 to 7 and 9 to 14 below(eg, selected from Table 13 or Table 14), eg, the second gene segment isa polymorphic variant of VH1-69.

Third Configuration an Isolated Antibody Having

(a) a heavy chain encoded by a nucleotide sequence produced followingrecombination in a transgenic non-human vertebrate cell of anunrearranged human immunoglobulin V gene segment with a human D andhuman J segment, optionally with affinity maturation in said cell,wherein one of the gene segments is derived from the genome of anindividual from a first human ethnic population; and the other two genesegments are derived from the genome of an individual from a second,different, human ethnic population, and wherein the antibody comprisesheavy chain constant regions of said non-human vertebrate (eg, rodent,mouse or rat heavy chain constant regions); and/or

(b) a light chain encoded by a nucleotide sequence produced followingrecombination in a transgenic non-human vertebrate cell of anunrearranged human immunoglobulin V gene segment with a human J segment,optionally with affinity maturation in said cell, wherein one of thegene segments is derived from the genome of an individual from a firsthuman ethnic population (optionally the same as the first population in(a)); and the other gene segment is derived from the genome of anindividual from a second, different, human ethnic population (optionallythe same as the second population in (a)), and wherein the antibodycomprises light chain constant regions of said non-human vertebrate (eg,rodent, mouse or rat heavy light constant regions);

(c) Optionally wherein each variable domain of the antibody is a humanvariable domain.

(d) Optionally wherein the heavy chain constant regions are gamma-typeconstant regions.

The invention also provides an isolated nucleotide sequence encoding theantibody, optionally wherein the sequence is provided in an antibodyexpression vector, optionally in a host cell.

The invention also provides a method of producing a human antibody, themethod comprising replacing the non-human vertebrate constant regions ofthe antibody of the third configuration with human antibody constantregions.

The invention also provides a pharmaceutical composition comprising anantibody according to the third configuration, or an antibody producedaccording to the method above and a diluent, excipient or carrier;optionally wherein the composition is provided in a container connectedto an IV needle or syringe or in an IV bag.

The invention also provides an antibody-producing cell that expressesthe second antibody recited in any one of the configurations.

In an alternative configuration, the invention contemplates thecombination of nucleotide sequences of first and second immunoglobulingene segments (eg, two or more polymorphic variants of a particularhuman germline VH or VL gene segment) to provide a synthetic genesegment. Such synthetic gene segment is used, in one embodiment, tobuild a transgenic immunoglobulin locus, wherein the synthetic genesegment is provided in combination with one or more human variable and Jregions (and optionally one or more human D regions) operably connectedupstream of a constant region. When provided in the genome of anon-human vertebrate or cell (eg, mouse or rat cell, eg, ES cell), theinvention provides for superhuman gene segment diversity. The sequencesto be combined can be selected from gene segments that have beenobserved to be commonly used in human antibodies raised against aparticular antigen (eg, a flu antigen, such as haemaglutinin). Bycombining the sequences, the synthetic gene segment may recombine invivo to produce an antibody that is well suited to the treatment and/orprevention of a disease or condition (eg, influenza) mediated by saidantigen.

Fourth Configuration

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises an immunoglobulin heavy chain locus comprisinghuman gene segment JH6*02, one or more VH gene segments and one or moreD gene segments upstream of a constant region; wherein the gene segmentsin the heavy chain locus are operably linked to the constant regionthereof so that the mouse is capable of producing an antibody heavychain produced by recombination of the human JH6*02 with a D segment anda VH segment.

A non-human vertebrate cell (optionally a mouse cell or a rat cell)whose genome comprises an immunoglobulin heavy chain locus comprisinghuman gene segment JH6*02, one or more VH gene segments and one or moreD gene segments upstream of a constant region; wherein the gene segmentsin the heavy chain locus are operably linked to the constant regionthereof for producing (eg, in a subsequent progeny cell) an antibodyheavy chain produced by recombination of the human JH6*02 with a Dsegment and a VH segment.

A heavy chain (eg, comprised by an antibody) isolated from a vertebrateof the invention wherein the heavy chain comprises a HCDR3 of at least20 amino acids.

A method for producing a heavy chain, VH domain or an antibody specificto a target antigen, the method comprising immunizing a non-humanvertebrate according to the invention with the antigen and isolating theheavy chain, VH domain or an antibody specific to a target antigen or acell producing the heavy chain, VH domain or an antibody, wherein theheavy chain, VH domain or an antibody comprises a HCDR3 that is derivedfrom the recombination of human JH6*02 with a VH gene segment and a Dgene segment.

A heavy chain, VH domain or an antibody produced by the method.

A B-cell or hybridoma expressing a heavy chain VH domain that isidentical to the VH domain of the heavy chain.

A nucleic acid encoding the VH domain of the heavy chain, or encodingthe heavy chain.

A vector (eg, a CHO cell or HEK293 cell vector) comprising the nucleicacid; optionally wherein the vector is in a host cell (eg, a CHO cell orHEK293 cell).

A pharmaceutical composition comprising the antibody, heavy chain or VHdomain (eg, comprised by an antibody), together with apharmaceutically-acceptable excipient, diluent or a medicament (eg, afurther antigen-specific variable domain, heavy chain or antibody).

The antibody, heavy chain or VH domain (eg, comprised by an antibody) asabove for use in medicine.

The use of an antibody, heavy chain or VH domain (eg, comprised by anantibody) as above in the manufacture of a medicament for treatingand/or preventing a medical condition in a human.

Fifth Configuration

A method of producing an antibody heavy chain, the method comprising

(a) providing an antigen-specific heavy chain variable domain; and

(b) combining the variable domain with a human heavy chain constantregion to produce an antibody heavy chain comprising (in N- toC-terminal direction) the variable domain and the constant region;

wherein

the human heavy chain constant region is an IGHG1 ref, IGHG2ref, IGHG2a,IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant region.

An antibody comprising a human heavy chain, the heavy chain comprising avariable domain that is specific for an antigen and a constant regionthat is an IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b,IGHG4ref or IGHG4a constant region. Optionally, the variable domaincomprises mouse-pattern AID somatic mutations.

A polypeptide comprising (in N- to C-terminal direction) a leadersequence, a human variable domain that is specific for an antigen and ahuman constant region that is an IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref,IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant region wherein (i) theleader sequence is not the native human variable domain leader sequence;and/or (ii) the variable domain comprises mouse AID-pattern somaticmutations and/or mouse Terminal deoxynucleotidyl transferase(TdT)-pattern junctional mutations.

A nucleotide sequence encoding (in 5′ to 3′ direction) a leader sequenceand a human antibody heavy chain, the heavy chain comprising a variabledomain that is specific for an antigen and a constant region that is anIGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4aconstant region; and the leader sequence being operable for expressionof the heavy chain and wherein the leader sequence is not the nativehuman variable domain leader sequence.

A nucleotide sequence encoding (in 5′ to 3′ direction) a promoter and ahuman antibody heavy chain, the heavy chain comprising a variable domainthat is specific for an antigen and a constant region that is anIGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4aconstant region; and the promoter being operable for expression of theheavy chain and wherein the promoter is not the native human promoter.

A vector (eg, a CHO cell or HEK293 cell vector) comprising a IGHG1ref,IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constantregion nucleotide sequence that is 3′ of a cloning site for theinsertion of a human antibody heavy chain variable domain nucleotidesequence, such that upon insertion of such a variable domain sequencethe vector comprises (in 5′ to 3′ direction) a promoter, a leadersequence, the variable domain sequence and the constant region sequenceso that the vector is capable of expressing a human antibody heavy chainwhen present in a host cell.

Sixth Configuration

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 3human variable region gene segments of the same type (eg, at least 3human VH6-1 gene segments, at least 3 human JH6 gene segments, at least3 human VK1-39 gene segments, at least 3 human D2-2 gene segments or atleast 3 human JK1 gene segments), wherein at least two of the human genesegments are variants that are not identical to each other.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different non-endogenous variable region gene segments of the same type(eg, at least 2 human VH6-1 gene segments, at least 3 human JH6 genesegments, at least 2 human VK1-39 gene segments, at least 2 human D2-2gene segments or at least 2 human JK1 gene segments) cis at the same Iglocus.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different human variable region gene segments of the same type (eg, atleast 2 human VH6-1 gene segments, at least 2 human JH6 gene segments,at least 2 human VK1-39 gene segments, at least 2 human D2-2 genesegments or at least 2 human JK1 gene segments) trans at the same Iglocus; and optionally a third human gene segment of the same type,wherein the third gene segment is cis with one of said 2 different genesegments.

A population of non-human vertebrates (eg, mice or rats) comprising arepertoire of human variable region gene segments, wherein the pluralitycomprises at least 2 human variable region gene segments of the sametype (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6gene segments, at least 2 human VK1-39 gene segments, at least 2 humanD2-2 gene segments or at least 2 human JK1 gene segments), a first ofsaid different gene segments is provided in the genome of a firstvertebrate of the population, and a second of said different genesegments being provided in the genome of a second vertebrate of thepopulation, wherein the genome of the first vertebrate does not comprisethe second gene segment.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different non-endogenous variable region gene segments of the same type(eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 genesegments, at least 2 human VK1-39 gene segments, at least 2 human D2-2gene segments or at least 2 human JK1 gene segments), wherein the genesegments are derived from the genome sequence of different humanindividuals that are not genetically related over at least 3generations.

A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 3 humanvariable region gene segments of the same type (eg, at least 3 humanVH6-1 gene segments, at least 3 human JH6 gene segments, at least 3human VK1-39 gene segments, at least 3 human D2-2 gene segments or atleast 3 human JK1 gene segments), wherein at least two of the human genesegments are variants that are not identical to each other.

A method of enhancing the immunoglobulin gene diversity of a non-humanvertebrate (eg, a mouse or rat), the method comprising providing thevertebrate with a genome comprising at least 2 different non-endogenousvariable region gene segments of the same type (eg, at least 2 humanVH6-1 gene segments, at least 2 human JH6 gene segments, at least 2human VK1-39 gene segments, at least 2 human D2-2 gene segments or atleast 2 human JK1 gene segments) cis at the same Ig locus.

A method of enhancing the immunoglobulin gene diversity of a non-humanvertebrate (eg, a mouse or rat), the method comprising providing thevertebrate with a genome comprising at least 2 different human variableregion gene segments of the same type (eg, at least 2 human VH6-1 genesegments, at least 2 human JH6 gene segments, at least 2 human VK1-39gene segments, at least 2 human D2-2 gene segments or at least 2 humanJK1 gene segments) trans at the same Ig locus; and optionally a thirdhuman gene segment of the same type, wherein the third gene segment iscis with one of said 2 different gene segments.

A method of providing an enhanced human immunoglobulin variable regiongene segment repertoire, the method comprising providing a population ofnon-human vertebrates (eg, a mouse or rat) comprising a repertoire ofhuman variable region gene segments, wherein the method comprisesproviding at least 2 different human variable region gene segments ofthe same type (eg, at least 2 human VH6-1 gene segments, at least 2human JH6 gene segments, at least 2 human VK1-39 gene segments, at least2 human D2-2 gene segments or at least 2 human JK1 gene segments),wherein a first of said different gene segments is provided in thegenome of a first vertebrate of the population, and a second of saiddifferent gene segments is provided in the genome of a second vertebrateof the population, wherein the genome of the first vertebrate does notcomprise the second gene segment.

A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous variable region gene segments of the same type (eg, atleast 2 human VH6-1 gene segments, at least 2 human JH6 gene segments,at least 2 human VK1-39 gene segments, at least 2 human D2-2 genesegments or at least 2 human JK1 gene segments), wherein the genesegments are derived from the genome sequence of different humanindividuals that are not genetically related over at least 3generations.

A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 humanvariable region gene segments of the same type (eg, at least 2 humanVH6-1 gene segments, at least 2 human JH6 gene segments, at least 2human VK1-39 gene segments, at least 2 human D2-2 gene segments or atleast 2 human JK1 gene segments), wherein the gene segments are derivedfrom the genome sequence of different human individuals that are notgenetically related over at least 3 generations; optionally wherein atleast 2 or 3 of said different gene segments are provided at the same Iglocus in said genome.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising first andsecond human Ig locus gene segments of the same type (eg, first andsecond human JH6 gene segments; or first and second IgG2 gene segments;or first and second human Jλ7 gene segments), wherein the first genesegment is a gene segment selected from any one of Tables 1 and 9 to 14(eg, selected from Table 13 or Table 14) (eg, IGHJ6-a) and the secondgene segment is the corresponding reference sequence.

A population of non-human vertebrates (eg, mice or rats) comprisingfirst and second human Ig locus gene segments of the same type (eg,first and second human JH6 gene segments; or first and second IgG2 genesegments; or first and second human Jλ7 gene segments), wherein thefirst gene segment is a gene segment selected from any one of Tables 1and 9 to 14 (eg, selected from Table 13 or Table 14) (eg, IGHJ6-a) andthe second gene segment is the corresponding reference sequence, whereinthe first gene segment is provided in the genome of a first vertebrateof the population, and the second gene segment is provided in the genomeof a second vertebrate of the population.

A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising first and second humanIg locus gene segments of the same type (eg, first and second human JH6gene segments; or first and second IgG2 gene segments; or first andsecond human Jλ7 gene segments), wherein the first gene segment is agene segment selected from any one of Tables 1 and 9 to 14 (eg, selectedfrom Table 13 or Table 14) (eg, IGHJ6-a) and the second gene segment isthe corresponding reference sequence.

In one aspect of this configuration, the invention relates to human Dgene segment variants as described further below.

In one aspect of this configuration, the invention relates to human Vgene segment variants as described further below.

In one aspect of this configuration, the invention relates to human Jgene segment variants as described further below.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 3: Schematic illustrating a protocol for producingrecombineered BAC vectors to add V gene segments into a mouse genome;

FIG. 4: Schematic illustrating a protocol for adding V gene segments toa mouse genome using sequential recombinase mediated cassette exchange(sRMCE); and

FIG. 5 (in 4 parts): Alignment of 13 IGHV1-69 variants showing thevariable (V) coding region only. Nucleotides that differ from VH1-69variant *01 are indicated at the appropriate position whereas identicalnucleotides are marked with a dash. Where nucleotide changes result inamino acid differences, the encoded amino acid is shown above thecorresponding triplet. Boxed regions correspond to CDR1, CDR2 and CDR3as indicated.

FIG. 6 is a schematic illustrating gene segment diversity and the effectof including variant variants in cis according to the invention:—

(a) Situation in a normal person: Recombination on the same chromosomelimits combinations of variants, for instance the antibody gene V4-4 canonly be recombined within variant 1 to form for instance for instanceV4-4-D-J6 or V4-4-D-J2A. Similarly the variant V4-4A can't be recombinedwith either J6 or J2A from variant 1 and can only be joined with J-genesfrom variant 2 to form V4-4A-D-J6A and V4-4A-D-J2. V4-4-J2/J6complexity=4.

(b) Situation in a transgenic mouse: Only one variant is provided so thegenome is limited. V4-4-J6/J2 complexity=2.

(c) Supra mouse of the invention: The variants are added in cis and thuscan be recombined in every combination, expanding the repertoire. Forinstance V4-4 can be combined with J6A, J6, J2A or J2 and similarlyV4-4A can be recombined with these same J-genes. The V4-4-J6/J2complexity=8, which in this simple example is double that of a personand 4× that of a mouse with a single variant.

FIG. 7: Alignment of human JH6*02 variants. Nucleotides that differ fromJH6 *01 are indicated at the appropriate position whereas identicalnucleotides are marked with a dash. Where nucleotide changes result inamino acid differences, the encoded amino acid is shown above. Accessionnumbers (eg, J00256) are shown to the left of the IMGT variant name.

FIG. 8: Alignment of JH sequences from various species.

FIG. 9: Codon Table

FIG. 10: BAC database extract

BRIEF DESCRIPTION OF THE TABLES

Table 1: Human IgH V Polymorphic Variants

Table 2: Human IgH D Polymorphic Variants

Table 3: Human IgH J Polymorphic Variants

Table 4: Human Ig Vk Polymorphic Variants

Table 5: Human Ig VA Polymorphic Variants

Table 6: Human IgH Jk Polymorphic Variants

Table 7: Human IgH Jλ Polymorphic Variants

Table 8: 1000 Genomes Project Human Populations

Table 9: Immunoglobulin Gene Usage in Human Antibody Responses toInfectious Disease Pathogens

Table 10A: Human IgH JH5 Variant Occurrences

Table 10B: Non-Synonymous Human IgH JH5 Variants

Table 11A: Human IgH JH6 Variant Occurrences

Table 11B: Non-Synonymous Human IgH JH6 Variants

Table 12A: Human IgH JH2 Variant Occurrences

Table 12B: Non-Synonymous Human IgH JH2 Variants

Table 13: Variant Frequency Analyses & Human Population Distributions

Table 14: Frequent Human Variant Distributions

Table 15: Human Gene Segment Usage: Heavy Chain Repertoires From NaiveNon-Human Vertebrates

Table 16: Human Gene Segment Usage: Heavy Chain Repertoires FromImmunised Non-Human Vertebrates

Table 17: Human Gene Segment Usage: Heavy Chain Repertoires FromAntigen-Specific Hybridomas

Table 18: Sequence Correlation Table

Table 19: Summary Of Function Correlated With Human Gamma ConstantRegion Sub-Type

Table 20: Gene Segments Prevalent In Few Human Populations

Table 21: Genomic and sequence information

DETAILED DESCRIPTION OF THE INVENTION

A suitable source of JH6*02 and other human DNA sequences for use in theinvention will be readily apparent to the skilled person. For example,it is possible to collect a DNA sample from a consenting human donor(eg, a cheek swab sample as per the Example herein) from which can beobtained suitable DNA sequences for use in constructing a locus of theinvention. Other sources of human DNA are commercially available, aswill be known to the skilled person. Alternatively, the skilled personis able to construct gene segment sequence by referring to one or moredatabases of human Ig gene segment sequences disclosed herein.

An example source for human V, D and J gene segments according to theinvention are Bacterial Artificial Chromosomes (RPCI-11 BACs) obtainedfrom Roswell Park Cancer Institute (RPCI)/Invitrogen. Seehttp://bacpac.chori.org/hmalell.htm, which describes the BACs asfollows:—

“RPCI-11 Human Male BAC Library

The RPCI-11 Human Male BAC Library (Osoegawa et al., 2001) wasconstructed using improved cloning techniques (Osoegawa et al., 1998)developed by Kazutoyo Osoegawa. The library was generated by KazutoyoOsoegawa. Construction was funded by a grant from the National HumanGenome Research Institute (NHGRI, NIH) (#1R01RG01165-03). This librarywas generated according to the new NHGRI/DOE “Guidance on Human Subjectsin Large-Scale DNA Sequencing . . . .

“Male blood was obtained via a double-blind selection protocol. Maleblood DNA was isolated from one randomly chosen donor (out of 10 maledonors)”.

-   Osoegawa K, Mammoser A G, Wu C, Frengen E, Zeng C, Catanese J J, de    Jong P J; Genome Res. 2001 March; 11(3):483-96; “A bacterial    artificial chromosome library for sequencing the complete human    genome”;-   Osoegawa, K., Woon, P. Y., Zhao, B., Frengen, E., Tateno, M.,    Catanese, J. J, and de Jong, P. J. (1998); “An Improved Approach for    Construction of Bacterial Artificial Chromosome Libraries”; Genomics    52, 1-8.

Superhuman Immunoglobulin Gene Repertoires

The invention relates to synthetically-extended & ethnically-diversesuperhuman immunoglobulin gene repertoires. The human immunoglobulinrepertoires are beyond those found in nature (ie, “Superhuman”), forexample, they are more diverse than a natural human repertoire or theycomprise combinations of human immunoglobulin gene segments fromdisparate sources in a way that is non-natural. Thus, the repertoires ofthe invention are “superhuman” immunoglobulin repertoires, and theinvention relates to the application of these in transgenic cells andnon-human vertebrates for utility in producing chimaeric antibodies(with the possibility of converting these into fully-human, isolatedantibodies using recombinant DNA technology). The present invention thusprovides for novel and potentially expanded synthetic immunoglobulindiversities, which provides for a pool of diversity from which antibodytherapeutic leads (antibody therapeutics and antibody tool reagents) canbe selected. This opens up the potential of transgenic human-mouse/rattechnologies to the possibility of interrogating different and possiblylarger antibody sequence-spaces than has hitherto been possible. To thisend, in one embodiment, the invention provides a SUPERHUMAN MOUSE™ (akaSUPRA-MOUSE™) and a SUPERHUMAN RAT™ (aka SUPRA-RAT™)

In developing this thinking, the present inventors have realised thepossibility of mining the huge genetics resources now available to theskilled person thanks to efforts such as the HapMap Project, 1000Genomes Project and sundry other immunoglobulin gene databases (seebelow for more details). Thus, in some embodiments, the inventorsrealised the application of these genome sequencing developments in thepresent invention to generate synthetically-produced andethnically-diverse artificial immunoglobulin gene repertoires. In oneaspect, the inventors realised that such repertoires are useful for theproduction of antibodies having improved affinity and/or biophysicalcharacteristics, and/or wherein the range of epitope specificitiesproduced by means of such repertoire is novel, provides for antibodiesto epitopes that have hitherto been intractable by prior transgenicimmunoglobulin loci or difficult to address.

The present invention provides libraries, vertebrates and cells, such astransgenic mice or rats or transgenic mouse or rat cells. Furthermore,the invention relates to methods of using the vertebrates to isolateantibodies or nucleotide sequences encoding antibodies. Antibodies,nucleotide sequences, pharmaceutical compositions and uses are alsoprovided by the invention.

Variation Analysis

The present inventors have realized methods and antibody loci designsthat harness the power of genetic variation analysis. The referencehuman genome provides a foundation for experimental work and geneticanalysis of human samples. The reference human is a compilation of thegenomes from a small number of individuals and for any one segment ofthe genome a high quality single reference genome for one of the twochromosomes is available. Because the reference genome was assembledfrom a series of very large insert clones, the identity of these clonesis known. Accordingly, experimental work with human genomic DNA isusually conducted on the clones from which the reference sequence wasderived.

Individual humans differ in their sequence and recently severalindividuals have had their genomes sequenced, for instance James Watsonand Craig Venter. Comparison of the genome sequence of these individualshas revealed differences between their sequences and the referencegenome in both coding and non-coding parts of the genome, approximately1 in 1000 bases are different. Some variants will be significant andcontribute to differences between individuals. In extreme cases thesewill result in genetic disease. Variation can be implicated in differingresponses to drugs administered to human patients, eg, yielding anundesirable lowering of patient response to treatment.

The 1000-Genomes Project has the objective of identifying the mostfrequent variations in the human genome. This public domain projectinvolved sequencing the genomes of more than 1000 individuals fromdiverse ethnic groups, comparing these sequences to the reference andassembling a catalogue of variants. This has enabled the annotation ofvariants in coding regions, but because this sequence wasn't derivedfrom large clones of DNA, the analysis of the sequence from diploidindividuals can't discriminate the distribution of the variation betweenthe maternal and paternally inherited chromosomes. Where more than onevariant is identified in a protein coding gene, it is not possible toilluminate the distribution of the pattern of variants in each versionof the protein. For example, if two variants are detected in differentpositions of the same protein in an individual, this could have resultedfrom one copy with two variants and none in the other or each copy couldhave just one variant. To illuminate the sequence of real proteins, the1000-Genome Project has sequenced mother-father-child trios. This allowsone to “phase” the sequence variants, in other words identify blocks ofsequence that are inherited from one or other parent and deconvolute thevariants.

To further understand the variation within the 1000-genome set a toolhas been developed that can identify the significant variants (definedas non-synonymous amino acid changes) from a region of DNA from thephased data in the 1000-genome data set. This tool has been madeavailable online http://www.1000genomes.org/variation-pattern-finder.This tool allows an investigator to download non-synonymous variationdelimited between specific coordinates. The downloaded files areconfigured as individual genotypes, but the data is phased so thehaplotype information and the frequencies of specific halotypes indifferent populations can be extracted.

The inventors' analysis of the 1000-genome data for the individual humancoding segments of the C, V D and J genes from the heavy and lightchains reveals that there is significant variation in these segments.Individuals will usually have two different heavy chain alleles and alsodifferent light chain alleles at both kappa and lambda loci. Therepertoire of antibodies that can be generated from each allele will bedifferent. This variation will contribute to a better or differingimmune response to certain antigens.

Humanized mice that have hitherto been generated with immunoglobulinheavy and light chain loci contain just one type of immunoglobulinlocus. Even if these mice contain a full human heavy chain locus, thevariation will be less than contained in a typical human because onlyone set of C, V, D and J genes are available, while a typical humanwould have two sets.

The inventors have devised ways to improve on this limitation whenconstructing transgenic non-human vertebrates and cells for humanantibody and variable region production in vivo.

Mice can be generated with two different loci, each engineered to have adifferent repertoire of V, D and J segments. This could be in a singlemouse or two or more separate mouse strains and would be analogous to orbeyond the repertoire found in a normal human. The engineering of such amouse would go beyond the repertoire described in humanized mice to datewhich only have one set of alleles.

However, the inventors also realized that this also has limitations,because the different loci would not normally interact to shuffle V, Dand J variants between loci. This same limitation is also inherent in ahuman, thus this system does not utilize the advantage of recombiningvariants in all combinations.

To go beyond the normal repertoire in humans and take advantage ofcombinations of C, V, D and J variants the inventors decided, in oneembodiment, to provide these on the same chromosome in cis. See FIG. 6.These loci would be characterized by having more than the normal numberof J, D or V genes. For example n=6 for the J genes, but including oneJ6 variant and one J2 variant would increase this to n=8. This could becombined with additional variants for the D and V genes, for example. Bydetailed analysis of the 1000-Genomes database, the inventors havedevised a collection of candidate polymorphic human variant genesegments, eg, JH gene segments (eg, see the examples), that can be builtinto the design of transgenic heavy and light chain loci in mice forexpressing increasingly diverse and new, synthetic repertoires of humanvariable regions. Moreover, by utilizing naturally-occurring humanvariant gene segments, as per embodiments of the invention, thisaddresses compatibility with human patients since the inventors analysishas drawn out candidate variants that are naturally conserved andsometimes very prevalent amongst human ethnic populations. Additionallythis enables one to tailor the configurations of the invention toprovide for antibody-based drugs that better address specific humanethnic populations.

In an example according to any configuration of the invention, loci (andcells and vertebrates comprising these) are provided in which genesegments from different human populations are used. This is desirable toincrease antibody gene diversity to better address more diverse humanpatients. In an example, the gene segments are from first and seconddifferent human populations respectively, and thus the second genesegment is found in the second human population, but not so (or rarely)in the first human population. Rarely means, for example, that the genesegment is found in 5, 4, 3, 2, or 1 or zero individuals in the firstpopulation in the 1000 Genomes database. For example, the first genesegment may be shown as present in a first population by reference toTable 13 or 14 herein, the second gene segment may be shown as presentin the second population by reference to Table 13 and not in the firstpopulation. Optionally, the first gene segment may also be shown asbeing present in the second population by reference to Table 13 or 14.

In any configuration or aspect of the invention, where a V gene segmentis used, this may be used optionally with the native leader sequence.For example, use of genomic DNA (eg, from BACs as in the examples) willmean that the native leader will be used for each V gene segmentincorporated into the locus and genomes of the invention. In analternative, the skilled person may wish to inert a non-native leadersequence together with one or more of the V gene segments. Similarly, inany configuration or aspect of the invention, where a V gene segment isused, this may be used optionally with the native 5′ UTR sequence. Forexample, use of genomic DNA (eg, from BACs as in the examples) will meanthat the native 5′ UTR sequence will be used for each V gene segmentincorporated into the locus and genomes of the invention. In analternative, the skilled person may wish to exclude the native 5′ UTRsequence.

The Present Invention Provides, in a First Configuration

(a) Superhuman Heavy Chain Gene Repertoires

A non-human vertebrate or vertebrate cell (optionally an ES cell orantibody-producing cell) comprising a genome having a superhumanimmunoglobulin heavy chain human VH and/or D and/or J gene repertoire.

In one aspect the cell of the invention is an embryonic stem cell. Forexample, the ES cell is derived from the mouse C57BL/6N, C57BL/6J, 129S5or 129Sv strain. In one aspect the non-human vertebrate is a rodent,suitably a mouse, and cells of the invention, are rodent cells or EScells, suitably mouse ES cells. The ES cells of the present inventioncan be used to generate animals using techniques well known in the art,which comprise injection of the ES cell into a blastocyst followed byimplantation of chimaeric blastocystys into females to produce offspringwhich can be bred and selected for homozygous recombinants having therequired insertion. In one aspect the invention relates to a transgenicanimal comprised of ES cell-derived tissue and host embryo derivedtissue. In one aspect the invention relates to genetically-alteredsubsequent generation animals, which include animals having a homozygousrecombinants for the VDJ and/or VJ regions.

The natural human immunoglobulin gene segment repertoire consists of(see eg, www.imgt.org):—

VH: total—125; functional—41 DH: total—27; functional—23 JH: total—8;functional—6

Vk: total—77; functional—38 Jk: total—5; functional—5

V lambda: total—75; functional—31

J lambda: total—7; functional—5

In one embodiment, the vertebrate or cell genome comprises a transgenicimmunoglobulin heavy chain locus comprising a plurality of humanimmunoglobulin VH gene segments, one or more human D gene segments andone or more human J gene segments, wherein the plurality of VH genesegments consists of more than the natural human repertoire offunctional VH gene segments; optionally wherein the genome is homozygousfor said transgenic heavy chain locus.

In one embodiment of the vertebrate or cell, the VH gene repertoireconsists of a plurality of VH gene segments derived from the genomesequence of a first human individual, supplemented with one or moredifferent VH gene segments derived from the genome sequence of a second,different human individual. Optionally the D and J segments are derivedfrom the genome sequence of the first human individual. Optionally theVH gene segments from the genome sequence of the second individual areselected from the VH gene segments listed in Table 1, 13 or 14. In thisway, the locus provides a superhuman repertoire of D gene segments.

Optionally the individuals are not related. Individuals are “notrelated” in the context of any configuration or aspect of the invention,for example, if one of the individuals does not appear in a family treeof the other individual in the same generation or going back one, two,three or four generations. Alternatively, are not related, for example,if they do not share a common ancestor in the present generation orgoing back one, two, three or four generations.

In one embodiment of the vertebrate or cell, the transgenic locuscomprises more than 41 functional human VH gene segment species, andthus more than the natural human functional repertoire. Optionally thelocus comprises at least 42, 43, 44, 45, 46, 47, 48, 49 or 50 functionalhuman VH gene segment species (eg, wherein the locus comprises the fullfunctional VH repertoire of said first individual supplemented with oneor more VH gene segments derived from the genome sequence of the secondhuman individual and optionally with one or more VH gene segmentsderived from the genome sequence of a third human individual). In thisway, the locus provides a superhuman repertoire of VH gene segments thatis useful for generating a novel gene and antibody diversity for use intherapeutic and tool antibody selection.

In one embodiment of the vertebrate or cell, the transgenic locuscomprises a first VH gene segment derived from the genome sequence ofthe first individual and a second VH gene segment derived from thegenome sequence of the second individual, wherein the second VH genesegment is a polymorphic variant of the first VH gene segment. Forexample, the VH gene segments are polymorphic variants of VH1-69 asillustrated in the examples below. Optionally the locus comprises afurther polymorphic variant of the first VH gene segment (eg, a variantderived from the genome sequence of a third human individual). In thisway, the locus provides a superhuman repertoire of VH gene segments.

In one embodiment of the vertebrate or cell, the genome (alternativelyor additionally to the superhuman VH diversity) comprises a transgenicimmunoglobulin heavy chain locus comprising a plurality of humanimmunoglobulin VH gene segments, a plurality of human D gene segmentsand one or more human J gene segments, wherein the plurality of D genesegments consists of more than the natural human repertoire offunctional D gene segments. Optionally the genome is homozygous for saidtransgenic heavy chain locus.

In one embodiment of the vertebrate or cell, the D gene repertoireconsists of a plurality of D gene segments derived from the genomesequence of a (or said) first human individual, supplemented with one ormore different D gene segments derived from the genome sequence of a (orsaid) second, different human individual. Optionally the individuals arenot related. Optionally the J segments are derived from the genomesequence of the first human individual. Optionally the D gene segmentsfrom the genome sequence of the second individual are selected from theD gene segments listed in Table 2, 13 or 14. In this way, the locusprovides a superhuman repertoire of D gene segments.

In one embodiment of the vertebrate or cell, the transgenic locuscomprises more than 23 functional human D gene segment species;optionally wherein the locus comprises at least 24, 25, 26, 27, 28, 29,30 or 31 functional human D gene segment species (eg, wherein the locuscomprises the full functional D repertoire of said first individualsupplemented with one or more D gene segments derived from the genomesequence of the second human individual and optionally with one or moreD gene segments derived from the genome sequence of a third humanindividual). In this way, the locus provides a superhuman repertoire ofD gene segments.

In one embodiment of the vertebrate or cell, the transgenic locuscomprises a first D gene segment derived from the genome sequence of thefirst individual and a second D gene segment derived from the genomesequence of the second individual, wherein the second D gene segment isa polymorphic variant of the first D gene segment. Optionally the locuscomprises a further polymorphic variant of the first D gene segment (eg,a variant derived from the genome sequence of a third human individual).In this way, the locus provides a superhuman repertoire of D genesegments.

In one embodiment of the vertebrate or cell (alternatively oradditionally to the superhuman VH and/or JH diversity), the genomecomprises a (or said) transgenic immunoglobulin heavy chain locuscomprising a plurality of human immunoglobulin VH gene segments, one ormore human D gene segments and a plurality of human JH gene segments,wherein the plurality of J gene segments consists of more than thenatural human repertoire of functional J gene segments; optionallywherein the genome is homozygous for said transgenic heavy chain locus.

In one embodiment of the vertebrate or cell, the JH gene repertoireconsists of a plurality of J gene segments derived from the genomesequence of a (or said) first human individual, supplemented with one ormore different J gene segments derived from the genome sequence of a (orsaid) second, different human individual. Optionally the individuals arenot related.

Optionally D segments are derived from the genome sequence of the firsthuman individual. Optionally the J gene segments from the genomesequence of the second individual are selected from the J gene segmentslisted in Table 3 13 or 14. In this way, the locus provides a superhumanrepertoire of JH gene segments.

In one embodiment of the vertebrate or cell, the transgenic locuscomprises more than 6 functional human JH gene segment segments.Optionally the locus comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 functional human JH gene segments (eg, wherein the locus comprisesthe full functional JH repertoire of said first individual supplementedwith one or more JH gene segments derived from the genome sequence ofthe second human individual and optionally with one or more JH genesegments derived from the genome sequence of a third human individual).In this way, the locus provides a superhuman repertoire of JH genesegments.

In one embodiment of the vertebrate or cell, the transgenic locuscomprises a first JH gene segment derived from the genome sequence ofthe first individual and a second JH gene segment derived from thegenome sequence of the second individual, wherein the second JH genesegment is a polymorphic variant of the first JH gene segment.Optionally the locus comprises a further polymorphic variant of thefirst JH gene segment (eg, a variant derived from the genome sequence ofa third human individual). In this way, the locus provides a superhumanrepertoire of JH gene segments.

(b) Superhuman Light Chain Gene Repertoires

The first configuration of the invention also provides:—

A non-human vertebrate or vertebrate cell (optionally an ES cell orantibody-producing cell) comprising a genome having a superhumanimmunoglobulin light chain human VL gene repertoire. Optionally thevertebrate or cell comprises a heavy chain transgene according to aspect(a) of the first configuration. Thus, superhuman diversity is providedin both the heavy and light chain immunoglobulin gene segments in thecell and vertebrate. For example, the genome of the cell or vertebrateis homozygous for the heavy and light chain transgenes and endogenousantibody expression is inactivated. Such a vertebrate is useful forimmunisation with a predetermined antigen to produce one or moreselected antibodies that bind the antigen and have human variableregions resulting from recombination within the superhuman gene segmentrepertoire. This provides potentially for a novel antibody and genesequence space from which to select therapeutic, prophylactic and toolantibodies.

In one embodiment of aspect (b) of the first configuration, thevertebrate or cell genome comprises

(i) a transgenic immunoglobulin kappa light chain locus comprising aplurality of human immunoglobulin VK gene segments and one or more humanJ gene segments, wherein the plurality of VK gene segments consists ofmore than the natural human repertoire of functional VK gene segments;optionally wherein the genome is homozygous for said transgenic kappalight chain locus; and/or

(ii) a transgenic immunoglobulin lambda light chain locus comprising aplurality of human immunoglobulin VA gene segments and one or more humanJ gene segments, wherein the plurality of VA gene segments consists ofmore than the natural human repertoire of functional VA gene segments;optionally wherein the genome is homozygous for said transgenic lambdalight chain locus.

In this way, the locus provides a superhuman repertoire of VL genesegments. In one embodiment of the vertebrate or cell,

(i) the VK gene repertoire consists of a plurality of VK gene segmentsderived from the genome sequence of a first human individual,supplemented with one or more VK gene segments derived from the genomesequence of a second, different human individual; optionally wherein theindividuals are not related; optionally wherein the J segments arederived from the genome sequence of the first human individual; andoptionally wherein the VK gene segments from the genome sequence of thesecond individual are selected from the VK gene segments listed in Table4, 13 or 14; and

(i) the VA gene repertoire consists of a plurality of VA gene segmentsderived from the genome sequence of a first human individual,supplemented with one or more VA gene segments derived from the genomesequence of a second, different human individual; optionally wherein theindividuals are not related; optionally wherein the J segments arederived from the genome sequence of the first human individual; andoptionally wherein the VA gene segments from the genome sequence of thesecond individual are selected from the VA gene segments listed in Table5, 13 or 14.

In this way, the locus provides a superhuman repertoire of VL genesegments.

In one embodiment of the vertebrate or cell,

-   -   the kappa light transgenic locus comprises more than 38        functional human VK gene segment species; optionally wherein the        locus comprises at least 39, 40, 41, 42, 43, 44, 45, 46, 47 or        48 functional human VK gene segment species (eg, wherein the        locus comprises the full functional VK repertoire of said first        individual supplemented with one or more VK gene segments        derived from the genome sequence of the second human individual        and optionally with one or more VK gene segments derived from        the genome sequence of a third human individual); and    -   the lambda light transgenic locus comprises more than 31        functional human VA gene segment species; optionally wherein the        locus comprises at least 32, 33, 34, 35, 36, 37, 38, 39, 40 or        41 functional human VA gene segment species (eg, wherein the        locus comprises the full functional VA repertoire of said first        individual supplemented with one or more VA gene segments        derived from the genome sequence of the second human individual        and optionally with one or more VA gene segments derived from        the genome sequence of a third human individual).

In this way, the locus provides a superhuman repertoire of VL genesegments.

In one embodiment of the vertebrate or cell,

-   -   the kappa light transgenic locus comprises a first VK gene        segment derived from the genome sequence of the first individual        and a second VK gene segment derived from the genome sequence of        the second individual, wherein the second VK gene segment is a        polymorphic variant of the first VK gene segment; optionally        wherein the locus comprises a further polymorphic variant of the        first VK gene segment (eg, a variant derived from the genome        sequence of a third human individual); and    -   the lambda light transgenic locus comprises a first VA gene        segment derived from the genome sequence of the first individual        and a second VA gene segment derived from the genome sequence of        the second individual, wherein the second VA gene segment is a        polymorphic variant of the first VA gene segment; optionally        wherein the locus comprises a further polymorphic variant of the        first VA gene segment (eg, a variant derived from the genome        sequence of a third human individual).

In this way, the locus provides a superhuman repertoire of VL genesegments.

In one embodiment of the vertebrate or cell, the genome comprises a (orsaid) transgenic immunoglobulin light chain locus comprising a pluralityof human immunoglobulin VL gene segments and a plurality of human JLgene segments, wherein the plurality of J gene segments consists of morethan the natural human repertoire of functional J gene segments;optionally wherein the genome is homozygous for said transgenic heavychain locus.

In one embodiment of the vertebrate or cell,

(i) the JK gene repertoire consists of a plurality of JK gene segmentsderived from the genome sequence of a (or said) first human individual,supplemented with one or more JK gene segments derived from the genomesequence of a (or said) second, different human individual; optionallywherein the individuals are not related; optionally wherein the VKsegments are derived from the genome sequence of the first humanindividual; optionally wherein the JK gene segments from the genomesequence of the second individual are selected from the JK gene segmentslisted in Table 6, 13 or 14; and

(ii) the JK gene repertoire consists of a plurality of Jλ gene segmentsderived from the genome sequence of a (or said) first human individual,supplemented with one or more Jλ gene segments derived from the genomesequence of a (or said) second, different human individual; optionallywherein the individuals are not related; optionally wherein the VAsegments are derived from the genome sequence of the first humanindividual; optionally wherein the Jλ gene segments from the genomesequence of the second individual are selected from the Jλ gene segmentslisted in Table 7, 13 or 14.

In this way, the locus provides a superhuman repertoire of JL genesegments. In one embodiment of the vertebrate or cell,

(i) the transgenic light chain locus comprises more than 5 functionalhuman JK gene segment species; optionally wherein the locus comprises atleast 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 functional human JK genesegment species (eg, wherein the locus comprises the full functional JKrepertoire of said first individual supplemented with one or more JKgene segments derived from the genome sequence of the second humanindividual and optionally with one or more JK gene segments derived fromthe genome sequence of a third human individual); and/or

(i) the transgenic light chain locus comprises more than 5 functionalhuman Jλ gene segment species; optionally wherein the locus comprises atleast 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 functional human Jλ genesegment species (eg, wherein the locus comprises the full functional Jλrepertoire of said first individual supplemented with one or more Jλgene segments derived from the genome sequence of the second humanindividual and optionally with one or more Jλ gene segments derived fromthe genome sequence of a third human individual).

In this way, the locus provides a superhuman repertoire of JL genesegments.

In one embodiment of the vertebrate or cell,

(i) the kappa light transgenic locus comprises a first JK gene segmentderived from the genome sequence of the first individual and a second JKgene segment derived from the genome sequence of the second individual,wherein the second JK gene segment is a polymorphic variant of the firstJK gene segment; optionally wherein the locus comprises a furtherpolymorphic variant of the first JK gene segment (eg, a variant derivedfrom the genome sequence of a third human individual); and

(ii) the lambda light transgenic locus comprises a first Jλ gene segmentderived from the genome sequence of the first individual and a second Jλgene segment derived from the genome sequence of the second individual,wherein the second JK gene segment is a polymorphic variant of the firstJλ gene segment; optionally wherein the locus comprises a furtherpolymorphic variant of the first Jλ gene segment (eg, a variant derivedfrom the genome sequence of a third human individual).

In this way, the locus provides a superhuman repertoire of JL genesegments. Further aspects of the first configuration are describedbelow.

The Present Invention Provides, in a Second Configuration

A library of antibody-producing transgenic cells whose genomescollectively encode a repertoire of antibodies, wherein

(a) a first transgenic cell expresses a first antibody having a chain(eg, heavy chain) encoded by a first immunoglobulin gene, the genecomprising a first variable domain nucleotide sequence producedfollowing recombination of a first human unrearranged immunoglobulingene segment (eg, a VH);

(b) a second transgenic cell expresses a second antibody having a chain(eg, a heavy chain) encoded by a second immunoglobulin gene, the secondgene comprising a second variable domain nucleotide sequence producedfollowing recombination of a second human unrearranged immunoglobulingene segment (eg, a VH), the first and second antibodies beingnon-identical;

(c) the first and second gene segments are different and derived fromthe genome sequences of first and second human individuals respectively,wherein the individuals are different; and optionally not related;

(d) wherein the cells are non-human vertebrate (eg, mouse or rat) cells(eg, B-cells or hybridomas).

In one embodiment, the library is provided in vitro. In anotherembodiment, the library is provided in vivo by one or a plurality oftransgenic non-human vertebrates. For example, the or each vertebrate isaccording to any aspect of the first configuration of the invention.

In one embodiment, the library encodes an antibody repertoire of from 10to 109 antibodies, for example, 10, 20, 30, 40, 50, 100 or 1000 to 108;or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or1000 to 106; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30,40, 50, 100 or 1000 to 104 antibodies. In an example, library encodes anantibody repertoire of at least 103, 104, 105, 106, 107, 108, 109, or1010 antibodies.

The first variable domain nucleotide sequence is produced followingrecombination of the first human unrearranged immunoglobulin genesegment with one or more other immunoglobulin gene segments (forexample, human immunoglobulin gene segments). For example, where thefirst gene segment is a VH, the first variable domain nucleotidesequence (a VH domain) is produced following recombination of the VHwith a human D and JH segments in vivo, optionally with somatichypermutation, in the first transgenic cell or an ancestor thereof. Forexample, where the first gene segment is a VL, the first variable domainnucleotide sequence (a VL domain) is produced following recombination ofthe VL with a human JL segment in vivo, optionally with somatichypermutation, in the first transgenic cell or an ancestor thereof.

The second variable domain nucleotide sequence is produced followingrecombination of the second human unrearranged immunoglobulin genesegment with one or more other immunoglobulin gene segments (forexample, human immunoglobulin gene segments). For example, where thesecond gene segment is a VH, the second variable domain nucleotidesequence (a VH domain) is produced following recombination of the VHwith a human D and JH segments in vivo, optionally with somatichypermutation, in the second transgenic cell or an ancestor thereof. Forexample, where the second gene segment is a VL, the second variabledomain nucleotide sequence (a VL domain) is produced followingrecombination of the VL with a human JL segment in vivo, optionally withsomatic hypermutation, in the second transgenic cell or an ancestorthereof.

The first and second gene segments are respectively derived from genomesequences of first and second human individuals. In one example, such agene segment is isolated or cloned from a sample cell taken from saidindividual using standard molecular biology techniques as know to theskilled person. The sequence of the gene segment may be mutated (eg, bythe introduction of up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15nucleotide changes) prior to use in the present invention. In anotherexample, a gene segment is derived by identifying a candidate humanimmunoglobulin gene segment in a database (see guidance below) and anucleotide sequence encoding a gene segment for use in the presentinvention is made by reference (eg, to be identical or a mutant with upto 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotide changes to thereference sequence) to the database sequence. The skilled person will beaware of methods of obtaining nucleotide sequences by reference todatabases or by obtaining from cellular samples.

In one embodiment of the vertebrate, cell or library of anyconfiguration of the invention, the first and second human individualsare members of first and second ethnic populations respectively, whereinthe populations are different. This, therefore, provides for superhumangene diversity in transgenic loci, cells and vertebrates as per theinvention.

Human Populations

Optionally the ethnic populations are selected from those identified inthe 1000 Genomes Project of database. In this respect, see Table 8 whichprovides details of the ethnic populations on which the 1000 Genomesdatabase is based.

N A Rosenberg et al (Science 20 Dec. 2002: vol. 298 no. 5602 2342-2343)studied the genetic structure of human populations of differinggeographical ancestry. In total, 52 populations were sampled, thesebeing populations with:

African Ancestry

(Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers ofNiger-Kordofanian languages (Bantu, Yoruba or Mandenka populations),

Eurasian Ancestry

(European ancestry (Orcadian, Adygei, Basque, French, Russians,Italians, Sardinian, Tuscan), Middle Eastern ancestry (Mozabite,Bedouin, Druze, Palestinians),

Central/South Asian ancestry (Balochl, Brahul, Makrani, Sindhi, Pathan,Burusho, Hazara, Uygur, Kalash)),

East Asian Ancestry

(Han, Dal, Daur, Hezhen, Lahu, Miao, Orogen, She, Tujia, Tu, Xibo, Yi,Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ancestry(Melanesian, Papuan); or

Americas Ancestry

(Karitiana, Surui, Colombian, Maya, Pima).

The International HapMap Project, Nature, 2003 December 18;426(6968):789-96, discloses that goal of the HapMap Project: todetermine the common patterns of DNA sequence variation in the humangenome by determining the genotypes of one million or more sequencevariants, their frequencies and the degree of association between themin DNA samples from populations with ancestry from parts of Africa, Asiaand Europe. The relevant human populations of differing geographicalancestry include Yoruba, Japanese, Chinese, Northern European andWestern European populations. More specifically:—

Utah population with Northern or Western European ancestry (samplescollected in 1980 by the Centre d'Etude du Polymorphisme Humain (CEPH));population with ancestry of Yoruba people from Ibadan, Nigeria;population with Japanese ancestry; and population with ancestry of HanChinese from China.

The authors, citing earlier publications, suggest that ancestralgeography is a reasonable basis for sampling human populations.

A suitable sample of human populations from which the populations usedin the present invention are selected is as follows:—

(a) European Ancestry

(b) Northern European ancestry; Western European ancestry; Toscaniancestry; British ancestry, Finnish ancestry or Iberian ancestry.

(c) More specifically, population of Utah residents with Northern and/orWestern European ancestry; Toscani population in Italia; Britishpopulation in England and/or Scotland; Finnish population in Finland; orIberian population in Spain.

(a) East Asian Ancestry

(b) Japanese ancestry; Chinese ancestry or Vietnamese ancestry.

(c) More specifically, Japanese population in Tokyo, Japan; Han Chinesepopulation in Beijing, China; Chinese Dai population in Xishuangbanna;Kinh population in Ho Chi Minh City, Vietnam; or Chinese population inDenver, Colo., USA.

(a) West African Ancestry

(b) Yoruba ancestry; Luhya ancestry; Gambian ancestry; or Malawianancestry.

(c) More specifically, Yoruba population in Ibadan, Nigeria; Luhyapopulation in Webuye, Kenya; Gambian population in Western Division, TheGambia; or Malawian population in Blantyre, Malawi.

(a) Population of the Americas

(b) Native American ancestry; Afro-Caribbean ancestry; Mexican ancestry;Puerto Rican ancestry; Columbian ancestry; or Peruvian ancestry.

(c) More specifically, population of African Ancestry in Southwest US;population of African American in Jackson, Miss.; population of AfricanCaribbean in Barbados; population of Mexican Ancestry in Los Angeles,Calif.; population of Puerto Rican in Puerto Rico; population ofColombian in Medellin, Colombia; or population of Peruvian in Lima,Peru.

(a) South Asian Ancestry

(b) Ahom ancestry; Kayadtha ancestry; Reddy ancestry; Maratha; orPunjabi ancestry.

(c) More specifically, Ahom population in the State of Assam, India;Kayadtha population in Calcutta, India; Reddy population in Hyderabad,India; Maratha population in Bombay, India; or Punjabi population inLahore, Pakistan.

In any configuration of the invention, in one embodiment, each humanpopulation is selected from a population marked “(a)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(b)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(c)” above.

In one embodiment of the library of the vertebrate, cell or library ofthe invention, the first and second ethnic populations are selected fromthe group consisting of an ethnic population with European ancestry, anethnic population with East Asian, an ethnic population with WestAfrican ancestry, an ethnic population with Americas ancestry and anethnic population with South Asian ancestry.

In one embodiment of the library of the vertebrate, cell or library ofthe invention, the first and second ethnic populations are selected fromthe group consisting of an ethnic population with Northern Europeanancestry; or an ethnic population with Western European ancestry; or anethnic population with Toscani ancestry; or an ethnic population withBritish ancestry; or an ethnic population with Icelandic ancestry; or anethnic population with Finnish ancestry; or an ethnic population withIberian ancestry; or an ethnic population with Japanese ancestry; or anethnic population with Chinese ancestry; or an ethnic populationVietnamese ancestry; or an ethnic population with Yoruba ancestry; or anethnic population with Luhya ancestry; or an ethnic population withGambian ancestry; or an ethnic population with Malawian ancestry; or anethnic population with Native American ancestry; or an ethnic populationwith Afro-Caribbean ancestry; or an ethnic population with Mexicanancestry; or an ethnic population with Puerto Rican ancestry; or anethnic population with Columbian ancestry; or an ethnic population withPeruvian ancestry; or an ethnic population with Ahom ancestry; or anethnic population with Kayadtha ancestry; or an ethnic population withReddy ancestry; or an ethnic population with Maratha; or an ethnicpopulation with Punjabi ancestry.

In one embodiment of any configuration of the vertebrate, cell orlibrary of the invention, the human immunoglobulin gene segment derivedfrom the genome sequence of the second individual is low-frequency(optionally rare) within the second ethnic population. Optionally humanimmunoglobulin gene segment has a Minor Allele Frequency (MAF)(cumulative frequency) of between 0.5%-5%, optionally less than 0.5%, inthe second human population, eg, as in the 1000 Genomes database.

In one embodiment of any configuration of the vertebrate, cell orlibrary of the invention, the first variable region nucleotide sequenceis produced by recombination of the first human immunoglobulin genesegment with a first J gene segment and optionally a first D genesegment, wherein the first human immunoglobulin gene segment is a V genesegment and the V, D and J segments are derived from the first humanpopulation, optionally from the genome of one individual of the firsthuman population.

In one embodiment of the library of the vertebrate, cell or library ofthe invention, the second variable region nucleotide sequence isproduced by recombination of the second human immunoglobulin genesegment with a second J gene segment and optionally a second D genesegment, wherein the second human immunoglobulin gene segment is a Vgene segment derived from the second population and the D and/or Jsegments are derived from the first human population, optionally the Dand J gene segments being from the genome of one individual of the firsthuman population.

In one embodiment of the library of the vertebrate, cell or library ofthe invention, all of the D and J segments that have been recombinedwith the first and second V gene segments are D and J segments derivedfrom the first human population, optionally the D and J gene segmentsbeing from the genome of one individual of the first human population.

In one embodiment of the library, the second human immunoglobulin genesegment is a polymorphic variant of the first human immunoglobulin genesegment; optionally wherein the second gene segment is selected from thegroup consisting of a gene segment in any of Tables 1 to 7 and 9 to 14(eg, selected from Table 13 or 14).

In one embodiment of the library, the first and second humanimmunoglobulin gene segments are both (i) VH gene segments; (ii) Dsegments; (iii) J segments (optionally both JH segments, both JKsegments or both Ĵ segments); (iv) constant regions (optionally both agamma constant region, optionally both a C gamma-1 constant region); (v)CH1 regions; (vi) CH2 regions; or (vii) CH3 regions.

The library is, for example, a naive and optionally has a library sizeof from 10 or 102 to 109 cells. For example, from 10, 20, 30, 40, 50,100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10,20, 30, 40, 50, 100 or 1000 to 10 s; or 10, 20, 30, 40, 50, 100 or 1000to 105; or 10, 20, 30, 40, 50, 100 or 1000 to 104 cells.

The library has, for example, been selected against a predeterminedantigen and optionally has a library size of from 10 or 102 to 109cells. For example, from 10, 20, 30, 40, 50, 100 or 1000 to 108; or 10,20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or 1000to 10 s; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30, 40,50, 100 or 1000 to 104 cells.

In one embodiment of the library of the invention, said first and secondcells are progeny of first and second ancestor non-human vertebratecells respectively, wherein the first ancestor cell comprises a genomecomprising said first human immunoglobulin gene segment; and the secondancestor cell comprises a genome comprising said second humanimmunoglobulin gene segment.

The invention further provides a library of antibody-producingtransgenic cells whose genomes collectively encode a repertoire ofantibodies, wherein the library comprises the first and second ancestorcells described above.

The invention further provides a library of hybridoma cells produced byfusion of the library of the invention (eg, a B-cell library) withfusion partner cells and optionally has a library size of from 10 or 102to 109 cells. For example, from 10, 20, 30, 40, 50, 100 or 1000 to 108;or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or1000 to 10 s; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30,40, 50, 100 or 1000 to 104 cells. Production of hybridomas is well knownto the skilled person. Examples of fusion partners are SP2/0-g14(obtainable from ECACC), P3XS3-Ag8.S53 (obtainable from LGC Standards;CRL-1580), NS1 and NS0 cells. PEG fusion or electrofusion can be carriedout, as is conventional.

The Invention Provides, in a Third Configuration:—

An isolated antibody having

(a) a heavy chain encoded by a nucleotide sequence produced followingrecombination in a transgenic non-human vertebrate cell of anunrearranged human immunoglobulin V gene segment with a human D andhuman J segment, optionally with affinity maturation in said cell,wherein one of the gene segments (eg, VH) is derived from the genome ofan individual from a first human ethnic population; and the other twogene segments (eg, D and JH) are derived from the genome of anindividual from a second (eg, a second and third respectively),different, human ethnic population, and wherein the antibody comprisesheavy chain constant regions (eg, C gamma) of said non-human vertebrate(eg, rodent, mouse or rat heavy chain constant regions); and/or

(b) a light chain encoded by a nucleotide sequence produced followingrecombination in a transgenic non-human vertebrate cell of anunrearranged human immunoglobulin V gene segment with a human J segment,optionally with affinity maturation in said cell, wherein one of thegene segments (eg, VL) is derived from the genome of an individual froma first human ethnic population (optionally the same as the firstpopulation in (a)); and the other gene segment (eg, JL) is derived fromthe genome of an individual from a second, different, human ethnicpopulation (optionally the same as the second population in (a)), andwherein the antibody comprises light chain constant regions of saidnon-human vertebrate (eg, rodent, mouse or rat heavy light constantregions);

(c) Optionally wherein each variable domain of the antibody is a humanvariable domain.

(d) Optionally wherein the heavy chain constant regions are mu- orgamma-type constant regions.

The invention also provides an isolated nucleotide sequence encoding theantibody of the third configuration, optionally wherein the sequence isprovided in an antibody expression vector, optionally in a host cell.Suitable vectors are mammalian expression vectors (eg, CHO cell vectorsor HEK293 cell vectors), yeast vectors (eg, a vector for expression inPichia pastoris, or a bacterial expression vector, eg, a vector for E.coli expression.

The invention also provides a method of producing a human antibody, themethod comprising replacing the non-human vertebrate constant regions ofthe antibody of the third configuration with human antibody constantregions (eg, a C variant disclosed in table 13 or 18). The skilledperson will be aware of standard molecular biology techniques to dothis. For example, see Harlow, E. & Lane, D. 1998, 5th edition,Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press,Plainview, N.Y.; and Pasqualini and Arap, Proceedings of the NationalAcademy of Sciences (2004) 101:257-259 for standard immunisation.Joining of the variable regions of an antibody to a human constantregion can be effected by techniques readily available in the art, suchas using conventional recombinant DNA and RNA technology as will beapparent to the skilled person. See e.g. Sambrook, J and Russell, D.(2001, 3'd edition) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Lab. Press, Plainview, N.Y.).

In one embodiment, the method comprises further making a mutant orderivative of the antibody.

The invention also provides a pharmaceutical composition comprising anantibody according to the third configuration, or a human antibody ofthe invention and a diluent, excipient or carrier; optionally whereinthe composition is provided in a container connected to an IV needle orsyringe or in an IV bag.

The invention also provides an antibody-producing cell (eg, a mammaliancell, eg, CHO or HEK293; a yeast cell, eg, P pastoris; a bacterial cell,eg, E coli; a B-cell; or a hybridoma) that expresses the second antibodyof the third configuration or the isolated antibody of the invention.

The First Configuration of the Invention Also Provides:—

A non-human vertebrate or vertebrate cell (optionally an ES cell orantibody-producing cell) whose genome comprises a transgenicimmunoglobulin locus (eg, a heavy chain locus or a light chain locus),said locus comprising immunoglobulin gene segments according to thefirst and second human immunoglobulin gene segments (optionally Vsegments) described above in connection with the third configuration.The gene segments are operably connected upstream of an immunoglobulinconstant region; optionally wherein the genome is homozygous for saidtransgenic immunoglobulin locus.

Optionally the immunoglobulin locus comprises more than the naturalhuman complement of functional V gene segments; and/or

Optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional D gene segments; and/or

Optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional J gene segments.

In this way, a superhuman immunoglobulin gene repertoire is provided ina transgenic non-human vertebrate or vertebrate cell according to theinvention.

The First Configuration Also Provides:—

A transgenic non-human vertebrate (eg, a mouse or rat) or vertebratecell (optionally an ES cell or antibody-producing cell) whose genomecomprises a transgenic immunoglobulin locus comprising a plurality ofhuman immunoglobulin gene segments operably connected upstream of anon-human vertebrate constant region for the production of a repertoireof chimaeric antibodies, or chimaeric light or heavy chains, having anon-human vertebrate constant region and a human variable region;wherein the transgenic locus comprises one or more human immunoglobulinV gene segments, one or more human J gene segments and optionally one ormore human D gene segments, a first (optionally a V segment) of saidgene segments and a second (optionally a V segment) of said genesegments being different and derived from the genomes of first andsecond human individuals respectively, wherein the individuals aredifferent; and optionally not related;

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional V gene segments; and/or

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional D gene segments; and/or

optionally wherein the immunoglobulin locus comprises more than thenatural human complement of functional J gene segments.

In this way, a superhuman immunoglobulin gene repertoire is provided ina transgenic non-human vertebrate or vertebrate cell according to theinvention.

The First Configuration Also Provides:—

A transgenic non-human vertebrate (eg, a mouse or rat) or vertebratecell (optionally an ES cell or antibody-producing cell) whose genomecomprises first and second transgenic immunoglobulin loci, each locuscomprising a plurality of human immunoglobulin gene segments operablyconnected upstream of a non-human vertebrate constant region for theproduction of a repertoire of chimaeric antibodies, or chimaeric lightor heavy chains, having a non-human vertebrate constant region and ahuman variable region;

wherein (i) the first transgenic locus comprises one or more humanimmunoglobulin V gene segments, one or more human J gene segments andoptionally one or more human D gene segments, (ii) the second transgeniclocus comprises one or more human immunoglobulin V gene segments, one ormore human J gene segments and optionally one or more human D genesegments; and (iii) wherein a first (optionally a V) gene segment ofsaid first locus and a second (optionally a V) gene segment of saidsecond gene locus are different and derived from the genomes of firstand second human individuals respectively, wherein the individuals aredifferent; and optionally not related;

optionally wherein the first and second loci are on differentchromosomes (optionally chromosomes with the same chromosome number) insaid genome;

optionally wherein each immunoglobulin locus comprises more than thenatural human complement of functional V gene segments; and/or

optionally wherein each immunoglobulin locus comprises more than thenatural human complement of functional D gene segments; and/or

optionally wherein each immunoglobulin locus comprises more than thenatural human complement of functional J gene segments.

In this way, a superhuman immunoglobulin gene repertoire is provided ina transgenic non-human vertebrate or vertebrate cell according to theinvention.

In these embodiments of the first configuration, the immunoglobulin genesegments are optionally as described for the third configuration.

In these embodiments of the first configuration, the genome optionallycomprises a third immunoglobulin gene segment (optionally a V segment),the third gene segment being derived from a human individual that isdifferent from the individual from which the first (and optionally alsothe second) gene segment is derived; optionally wherein the first,second and third gene segments are polymorphic variants of a humanimmunoglobulin gene segment (eg, VH1-69—see the examples for furtherdescription).

In these embodiments of the first configuration, the genome of thevertebrate or cell is optionally homozygous for the first, second andoptional third gene segment, wherein a copy of the first, second andoptional third gene segments are provided together on the samechromosome operably connected upstream of a common non-human vertebrateconstant region.

For example, each first, second and optional third gene segment is a Vgene segment.

In one example, the library of the invention is provided by a collectionof non-human vertebrates (optionally a collection of rodents, mice orrats); optionally, wherein a first member of said collection producessaid first antibody but not said second antibody, and a second member ofthe collection produces said second antibody (but optionally not saidfirst antibody). It is therefore contemplated to make non-humanvertebrates where different human genomes have been used as a source forbuilding the transgenic loci in the vertebrates. For example, a firstvertebrate comprises a transgenic heavy chain locus having gene segmentsonly from a first (and optionally a second) human population orindividual; a second vertebrate comprises a transgenic heavy chain locushaving gene segments only from a third (and optionally a fourth) humanpopulation or individual; and optionally third and more vertebrates canbe built similarly based on unique or overlapping human populationgenomes. However, when provided as a mixed population of transgenicvertebrates, the mixed population provides a collective pool of humanimmunoglobulin genes that is greater than found in a natural humanrepertoire. This is useful to extend the antibody and gene sequencespace beyond those possible with prior transgenic mice and rats bearinghuman immunoglobulin loci. As explained above, these have been based ona single human genome.

In one embodiment, the collection of non-human vertebrates bear humanimmunoglobulin genes confined to human populations that are togethergrouped under the same population genus “(a)” mentioned above. Thisprovides for a gene repertoire that is biased to producing humanantibody variable regions prevalent in the population genus (a) and thususeful for generating antibody therapeutics/prophylactics for members ofsaid population. Alternatively, where gene segments from different humanpopulations are provided in a single transgene according to theinvention (not necessarily in a collection of vertebrates), thedifferent human populations are for example together grouped under thesame population genus “(a)” mentioned above.

The invention also provides a repertoire of antibodies expressed from alibrary of cells according to the invention.

In the non-human vertebrate or cell of any configuration of theinvention, the constant region of the transgenic locus is, in oneexample, an endogenous constant region of said vertebrate (eg,endogenous mouse or rat constant region, eg, from the same strain ofmouse or rat as the non-human vertebrate itself).

The invention also provides a method of constructing a cell (eg, an EScell) according to the invention, the method comprising

(a) identifying functional V and J (and optionally D) gene segments ofthe genome sequence of a (or said) first human individual;

(b) identifying one or more functional V and/or D and/or J gene segmentsof the genome sequence of a (or said) second human individual, whereinthese additional gene segments are not found in the genome sequence ofthe first individual;

(c) and constructing a transgenic immunoglobulin locus in the cell,wherein the gene segments of (a) and (b) are provided in the locusoperably connected upstream of a constant region.

Optionally the cell comprises a heavy chain locus constructed accordingto steps (a) to (c) and/or a light chain locus (kappa and/or lambdaloci) constructed according to steps (a) to (c).

Optionally the cell is homozygous for the or each transgenic locus;optionally wherein antibody expression from loci endogenous to said cellhas been inactivated. This is useful for confining the functionalantibody gene repertoire, and thus antibody production, to antibodiesbearing human variable regions.

Optionally the gene segment(s) in step (b) are identified from animmunoglobulin gene database selected from the 1000 Genomes, Ensembl,Genbank and IMGT databases.

Optionally the first and second human individuals are members of firstand second ethnic populations respectively, wherein the populations aredifferent, optionally wherein the human immunoglobulin gene segmentderived from the genome sequence of the second individual islow-frequency (optionally rare) within the second ethnic population.

The invention also provides a method of making a transgenic non-humanvertebrate (eg, a mouse or rat), the method comprising

(a) constructing an ES cell (eg, a mouse C57BL/6N, C57BL/6J, 129S5 or129Sv strain ES cell) by carrying out the method above;

(b) injecting the ES cell into a donor non-human vertebrate blastocyst(eg, a mouse C57BL/6N, C57BL/6J, 129S5 or 129Sv strain blastocyst);

(c) implanting the blastocyst into a foster non-human vertebrate mother(eg, a C57BL/6N, C57BL/6J, 129S5 or 129Sv strain mouse); and

(d) obtaining a child from said mother, wherein the child genomecomprises a transgenic immunoglobulin locus.

The invention provides a transgenic non-human vertebrate (eg, a mouse orrat) made by the method or a progeny thereof. The invention alsoprovides a population of such non-human vertebrates.

Microinjection of ES cells into blastocysts and generation of transgenicmice thereafter are conventional practices in the state of the art, andthe skilled person is aware of techniques useful to effect this.C57BL/6N, C57BL/6J, 129S5 or 129Sv mouse strains and ES cells arereadily and publicly available.

The invention also provides a method of isolating an antibody that bindsa predetermined antigen (eg, a bacterial or viral pathogen antigen), themethod comprising

(a) providing a vertebrate (optionally a mouse or rat) according to theinvention;

(b) immunising (eg, using a standard prime-boost method) said vertebratewith said antigen (optionally wherein the antigen is an antigen of aninfectious disease pathogen);

(c) removing B lymphocytes from the vertebrate and selecting one or moreB lymphocytes expressing antibodies that bind to the antigen;

(d) optionally immortalising said selected B lymphocytes or progenythereof, optionally by producing hybridomas therefrom; and

(e) isolating an antibody (eg, and IgG-type antibody) expressed by the Blymphocytes; and

(f) optionally producing a derivative or variant of the antibody.

This method optionally further comprises after step (e) the step ofisolating from said B lymphocytes nucleic acid encoding said antibodythat binds said antigen; optionally exchanging the heavy chain constantregion nucleotide sequence of the antibody with a nucleotide sequenceencoding a human or humanised heavy chain constant region and optionallyaffinity maturing the variable region of said antibody; and optionallyinserting said nucleic acid into an expression vector and optionally ahost.

Bioinformatics Analysis & Selection of Immunoglobulin Gene Segments

See also the discussion on variation analysis above.

The skilled person will know of sources of human antibody genesequences, such as IMGT (www.imgt.org), GenBank(www.ncbi.nlm.nih.gov/genbank) Bioinformatics tools for databasemanipulation are also readily available and known to the skilled person,eg, as publicly available from the 1000 Genomes Project/EBI(www.1000genomes.org)

As a source of antibody gene segment sequences, the skilled person willalso be aware of the following available databases and resources(including updates thereof):—

1.1. The Kabat Database (G. Johnson and T. T. Wu, 2002;http://www.kabatdatabase.com). Created by E. A. Kabat and T. T. Wu in1966, the Kabat database publishes aligned sequences of antibodies,T-cell receptors, major histocompatibility complex (MHC) class I and IImolecules, and other proteins of immunological interest. A searchableinterface is provided by the SeqhuntII tool, and a range of utilities isavailable for sequence alignment, sequence subgroup classification, andthe generation of variability plots. See also Kabat, E. A., Wu, T. T.,Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteinsof Immunological Interest, 5th ed., NIH Publication No. 91-3242,Bethesda, Md., which is incorporated herein by reference, in particularwith reference to human gene segments for use in the present invention.

1.2. KabatMan (A. C. R. Martin, 2002;http://www.bioinf.org.uk/abs/simkab.html). This is a web interface tomake simple queries to the Kabat sequence database.

1.3. IMGT, the International ImMunoGeneTics Information System®; M.-P.Lefranc, 2002; http://imgt.cines.fr). IMGT is an integrated informationsystem that specializes in antibodies, T cell receptors, and MHCmolecules of all vertebrate species. It provides a common portal tostandardized data that include nucleotide and protein sequences,oligonucleotide primers, gene maps, genetic polymorphisms,specificities, and two-dimensional (2D) and three-dimensional (3D)structures. IMGT includes three sequence databases (IMGT/LIGM-DB,IMGT/MHC-DB, IMGT/PRIMERDB), one genome database (IMGT/GENE-DB), one 3Dstructure database (IMGT/3Dstructure-DB), and a range of web resources(“IMGT Marie-Paule page”) and interactive tools.

1.4. V-BASE (I. M. Tomlinson, 2002; http://www.mrc-cpe.cam.ac.uk/vbase).V-BASE is a comprehensive directory of all human antibody germlinevariable region sequences compiled from more than one thousand publishedsequences. It includes a version of the alignment software DNAPLOT(developed by Hans-Helmar Althaus and Werner Muller) that allows theassignment of rearranged antibody V genes to their closest germline genesegments.

1.5. Antibodies—Structure and Sequence (A. C. R. Martin, 2002;http://www.bioinf.org.uk/abs). This page summarizes useful informationon antibody structure and sequence. It provides a query interface to theKabat antibody sequence data, general information on antibodies, crystalstructures, and links to other antibody-related information. It alsodistributes an automated summary of all antibody structures deposited inthe Protein Databank (PDB). Of particular interest is a thoroughdescription and comparison of the various numbering schemes for antibodyvariable regions.

1.6. AAAAA—AHo's Amazing Atlas of Antibody Anatomy (A. Honegger, 2001;http://www.unizh.ch/˜antibody). This resource includes tools forstructural analysis, modeling, and engineering. It adopts a unifyingscheme for comprehensive structural alignment of antibody andT-cell-receptor sequences, and includes Excel macros for antibodyanalysis and graphical representation.

1.7. WAM—Web Antibody Modeling (N. Whitelegg and A. R. Rees, 2001;http://antibody.bath.ac.uk). Hosted by the Centre for Protein Analysisand Design at the University of Bath, United Kingdom. Based on the AbMpackage (formerly marketed by Oxford Molecular) to construct 3D modelsof antibody Fv sequences using a combination of established theoreticalmethods, this site also includes the latest antibody structuralinformation.

1.8. Mike's Immunoglobulin Structure/Function Page (M. R. Clark, 2001;http://www.path.carmac.uk/˜mrc7/mikeimages.html) These pages provideeducational materials on immunoglobulin structure and function, and areillustrated by many colour images, models, and animations. Additionalinformation is available on antibody humanization and Mike Clark'sTherapeutic Antibody Human Homology Project, which aims to correlateclinical efficacy and anti-immunoglobulin responses with variable regionsequences of therapeutic antibodies.

1.9. The Antibody Resource Page (The Antibody Resource Page, 2000;http://www.antibodyresource.com). This site describes itself as the“complete guide to antibody research and suppliers.” Links to amino acidsequencing tools, nucleotide antibody sequencing tools, andhybridoma/cell-culture databases are provided.

1.9. Humanization bYDesign (J. Saldanha, 2000;http://people.cryst.bbk.ac.uk/˜ubcg07s). This resource provides anoverview on antibody humanization technology. The most useful feature isa searchable database (by sequence and text) of more than 40 publishedhumanized antibodies including information on design issues, frameworkchoice, framework back-mutations, and binding affinity of the humanizedconstructs.

See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo,Methods in Molecular Biology™, Human Press. Also athttp://www.blogsua.com/pdf/antibody-engineering-methods-and-protocolsantibody-engineering-methods-and-protocols.pdf

As a source of genomic sequence variation data, the skilled person willalso be aware of the following available databases and resources(including updates thereof):—

1. HapMap (The International HapMap Consortium. 2003;http://hapmap.ncbi.nlm.nih.gov/index.html.en). The HapMap Project is aninternational project that aims to compare the genetic sequences ofdifferent individuals to identify chromosomal regions containing sharedgenetic variants. The HapMap www site provides tools to identifychromosomal regions and the variant therein, with options to drill downto population level frequency data.

2. 1000 Genomes (The 1000 Genomes Project Consortium 2010;http://www.1000genomes.org/). This resource provides complete genomicsequence for 2500 unidentified individuals from one of 25 distinctpopulation groups, with the aim of identifying genomic variants of >1%.The site provides the ability to interrogate data utilizing online tools(e.g. Variation Pattern Finder) and to download variant data forindividual population groups.

3. Japanese SNP Database (H. Haga et al. 2002;http://snp.ims.u-tokyo.ac.jp/index.html). Based on a study identifying190,562 human genetic variants this site catalogues genomic variantswith useful features for searching and summarizing data.

It is possible to identify variants in immunoglobulin genes classed aslow-frequency or rare variants that segregate with specific human ethnicpopulations. For the purpose of this analysis, a low-frequencyimmunoglobulin gene segment is classed as one with ‘Minor AlleleFrequency’ (MAF) (cumulative frequency) of between 0.5%-5%, rarevariants are those classed as having a MAF of less than 0.5% in aparticular human population.

The following bioinformatics protocol is envisaged to identify humanimmunoglobulin gene segments for use in the present invention:

(a) Identify one or more genomic regions containing gene segments ofinterest (“target genomic regions”) and calculate the genomiccoordinates, using coordinates that match the sequence assembly buildused by either the 1000 Genomes project or International HapMap project(or another selected human gene database of choice).

(b) Identify genomic variants mapped to the genomic regions previouslyidentified in (a). Retrieve variant frequencies for variants for eachsuper population and preferably sub-population where such data isavailable. Tools readily available on the HapMap WWW site and the VWCtools for the 1000Genomes Project are useful for this step.

(c) Filter list of genomic variants from target genomic regions tocontain only variants classed as either ‘Non-synonymous’ singlenucleotide polymorphisms (SNPs) or genomic ‘insertions or defections’(indels). Filter further to include those that are present in exonicsequences only.

(d) Correlate population frequency data for each of the identifiedvariants for each of the super populations (for example ‘EuropeanAncestry’, ‘East Asian ancestry’, ‘West African ancestry’, ‘Americas’,and ‘South Asian ancestry’) to identify those variants that segregatewith less than two super-populations. Further correlate all identifiedvariants with each of the sub-populations (for example, ‘Europeanancestry’ super-population might be subdivided into groups such as‘CEU—Utah residents with Northern or Western European ancestry’, ‘TSIToscani in Italia’ and ‘British from England and Scotland’) and producea second score for rarity of variants in within a super-population.

(e) Collect one or more gene segments that show segregation to specificsub-populations for construction of synthetic loci according to theinvention.

In one embodiment throughout the present text, “germline” refers to thecanonical germline gene segment sequence.

By detailed analysis of the 1000 Genomes database, the inventors havedevised a collection of candidate polymorphic antibody gene segmentvariants, eg, human variant JH gene segments (eg, see Example 4), thatcan be built into the design of transgenic heavy chain loci in mice forexpressing increasingly diverse and new, synthetic repertoires of humanvariable regions. To this end, the invention provides the followingembodiments.

The Present Invention Provides in a Fourth Configuration—

Selection of Human JH6*02 Variant

Transgenic IgH Loci, Non-Human Vertebrates, Cells & Antibodies Based onHuman JH6*02

As explained above, in designing transgenic Ig heavy chain loci thepresent inventors have considered the huge amount of data available fromthe 1000 Genomes project (see www.1000genomes.org) that analyses genedistributions amongst many human populations, and in particular data onIg gene segments. The inventors were also aware of human gene segmentsdisclosed in the IMGT database (see www.imgt.org) and in Ensembl (seewww.ensembl.org). The inventors needed to make choices about which humangene segments to include amongst the large number of human gene segmentspresented in these databases and the other sources of human Ig genesegment information known in the art, including those other databasesdisclosed herein. When choosing human JH gene segments, the inventorswere aware that human JH6 encodes a relatively long amino acid sequence,and thus the inventors thought it desirable to include this forincreasing the chances of producing IgH chains with relatively longHCDR3 regions. Antibodies with long HCDR3 (at least 20 amino acidsaccording to IMGT nomenclature) have been shown to neutralise a varietyof pathogens effectively including HIV, Influenza virus, malaria andAfrica trypanosomes. Reference is also made to naturally-occurringCamelid (eg, llama or camel) heavy chain-only antibodies which bear longHCDR3s for reaching relatively inaccessible epitopes (see, eg,EP0937140). Long HCDR3s can form unique stable subdomains with extendedloop structure that towers above the antibody surface to confer finespecificity. In some cases, the long HCDR3 itself is sufficient forepitope binding and neutralization (Liu, L et al; Journal of Virology.2011. 85: 8467-8476, incorporated herein by reference). The uniquestructure of the long HCDR3 allows it to bind to cognate epitopes withininaccessible structure or extensive glycosylation on a pathogen surface.In human peripheral blood, there is around 3.5% of naive B antibodies or1.9% of memory B IgG antibodies containing the HCDR3s with lengths ofmore than 24 amino acids (PLoS One. 2012; 7(5):e36750. Epub 2012 May 9;“Human peripheral blood antibodies with long HCDR3s are establishedprimarily at original recombination using a limited subset of germlinegenes”; Briney B S e al, incorporated herein by reference) (FIG. 1). Theusage analysis indicates that these antibodies have the preference touse human JH6 with human D2-2, D3-3 or D2-15 (Brinley, B S et al, FIGS.2-5). See also PLoS One. 2011 March 30; 6(3):e16857; Comparison ofantibody repertoires produced by HIV-1 infection, other chronic andacute infections, and systemic autoimmune disease”; Breden F et al,incorporated herein by reference. Around 20% of all HCDR3 of antibodiesuse JH6. However, in those antibodies with HCDR3 of more than 24 aminoacids, 70% use JH6 (Brinley, B S et al, FIG. 2).

There is a need in the art for genetically modified non-humanvertebrates and cells that can make antibodies and heavy chains thathave long human HCDR3s, as well as antibodies, chains and VH domainsthat can be selected from such vertebrates and cells wherein these canaddress target epitopes better accessed by long HCDR3s.

The inventors, therefore, chose in this configuration of the inventionto include a human JH6 gene segment as a mandatory human gene segment intheir IgH locus design. Several different naturally-occurring human JH6variants are known (eg, JH6*01 to *04 as well as others; IMGTnomenclature). The inventors considered this when deciding upon whichhuman JH6 variant should be included in the transgenic IgH locus design.An alignment of some human JH6 variants is shown in FIG. 7 (fromwww.imgt.org; dashes indicate identical nucleotides; nucleotide changesversus the *01 variant are shown by underlined nucleotides andcorresponding amino acid changes are shown by underlined amino acids;Genbank accession numbers (release 185.0) are shown prefixed by J, X, Mor A). The inventors used sequencing of human genomic DNA samples,inspection of public IgH DNA databases as well as informed choices onthe basis of variant sequences as means to arrive at a rational choiceof which JH6 variant to use.

The 1000 Genomes database uses human JH6*03 as the reference sequence,which would be a possible choice for the skilled person wishing toconstruct a transgenic IgH locus. The inventors noticed (eg, FIG. 7herein) that position 6 in JH6*03 is a tyrosine (Y) encoded by a TACcodon, whereas some other naturally-occurring human variants have aglycine (G) encoded by a GGT codon (the glycine being present as a YYGmotif, forming part of a larger YYGXDX motif). To understand thepotential significance of this, the inventors carried out analysis of JHsequences from other vertebrate species. The inventors surprisinglynoticed that YYG and YYGXDX motifs are conserved across many vertebratespecies (see FIGS. 7 & 8). This suggested to the inventors, therefore,that preservation of this motif might be desirable, which could guidethe choice of JH6 variant for use in the present invention.

Another pointer arose when the inventors considered the TAC codon versusthe GGT codon encoding Y or G respectively. The inventors considered theimpact of these nucleotide sequences on the action of activation-inducedcytidine deaminase (AID). The inventors knew that activation-inducedcytidine deaminase (AID) is believed to initiate Ig somatichypermutation (SHM) in a multi-step mechanism and they addressed thisactivity when rationally designing the locus. AID catalyses thedeamination of C to U in DNA, generating mutations at C bases. Cytidineslocated within hotspot motifs are preferentially deaminated. Certainmotifs are hotspots for AID activity (DGYW, WRC, WRCY, WRCH, RGYW, AGY,TAC, WGCW, wherein W=A or T, Y=C or T, D=A, G or T, H=A or C or T, andR=A or G). The presence of a TAC codon encoding Y at position 6 inJH6*03 creates AID mutation hotspots (the cytidine being the substrateof AID), these hotspots being the underlined motifs in the previoussentence. The inventors considered the impact of this and in doing sothey considered possible mutants created by AID activity at thecytidine. Reference is made to FIG. 9. The inventors noticed that amutation at the third base of the TAC codon would yield 3 possibleoutcomes: Y, stop or stop. Thus, out of the three stop codons possiblein the genetic code (the other being encoded by TGA—see FIG. 9), two ofthem would be provided by mutation of the cytidine in the TAC codonencoding position 6 in JH6*03. The inventors, therefore, considered thatthis might increase the chances of non-productive IgH variable regionproduction in transgenic loci based on JH6*03. Moreover, the inventorsnoticed that provision of a GGT codon instead (as per the other humanJH6 variants) seemed preferable since mutation of the third base wouldnever yield a stop codon (see FIG. 9), and furthermore would retaincoding, and thus conservation, of glycine at position 6, which theinventors also noticed was is in the YYG and YYGXDX motifs conservedacross species.

Having decided against using JH6*03, the inventors needed to make achoice from other possible human variants. The MDV motif is at theC-terminus of HCDR3 based on human JH6, the adjacent framework 4 (FW4)starting with the WGQ motif (with reference to the sequence shownencoded by JH6*01; FIG. 7). In making their choices for locus design,the inventors wished to maximise conservation of this HCDR3/FW4 junctionin product IgH chains and antibodies including these. The inventorsbelieved this to be desirable for heavy chain variable domainfunctionality and conformation. The inventors thought that this might insome cases be desirable to minimise immunogenicity (suitable for humanpharmaceutical use). Consistent with these considerations, the inventorswanted to make a choice that would minimise mutation around theHCDR3/FW4 junction as a result of SHM in vivo to conserve junctionconfiguration. See Rogozin & Diaz; “Cutting Edge: DGYW/WRCH Is a BetterPredictor of Mutability at G:C Bases in Ig Hypermutation Than the WidelyAccepted RGYW/WRCY Motif and Probably Reflects a Two-StepActivation-Induced Cytidine Deaminase-Triggered Process”; Journal ofImmunology; Mar. 15, 2004 vol. 172 no. 6 3382-3384. An example of a DGYWmotif is GGCA. The inventors had this in mind when analysing the variantsequences.

With these considerations in mind, the inventors decided specifically touse human JH6*02 as the mandatory human JH6 for their IgH locus design.JH6*01 was rejected as the mandatory JH6 gene segment since thenucleotide sequence GGG Ĉ A (encoding G and Q) contains a GGCA motifwhich is an AID recognition hotspot. The inventors realised that JH6*04also contains such a motif due to the presence of the sequence GGC AAAencoding G and K (positions 11 and 12 respectively). The inventors alsorealised that the *02 variant has a C instead of a G that is in the *01variant, the C desirably being a synonymous change (ie, not changing theencoded amino acid sequence around the CDR3/FW4 junction) and also thisdoes not provide a GGCA AID hotspot motif. The inventors, therefore,decided that the mandatory JH6 should have this C base and this toopointed them to using the human JH6*02 variant.

In one example of any configuration of the invention herein, the onlyJH6 species included in the locus or genome is human JH6*02.

The inventors obtained 9 anonymised DNA samples from cheek swabs of 9consenting human adults. Sequencing was performed on IgH locus DNA toconfirm natural JH6 variant usage. It was found that the genome of all 9humans contained a JH6*02 variant gene segment. In 7 out of the 9humans, the genome was homozygous for JH6*02 (ie, each chromosome 14 hadJH6*02 as its JH6 gene segment in the IgH locus). The inventors alsoinspected the publicly-available sequence information from the genomesof well-known scientists Craig Venter and Jim Watson. Both of thesegenomes contain JH6*02 too. This indicated to the inventors that thisvariant is common in humans.

So, the inventors made a choice of human JH6*02 on the basis of

(i) Containing the YYG and YYGXDX motifs that is conserved acrossseveral vertebrate species;

(ii) Provision of one less TAC codon (an AID hotspot that risks stopcodons) and a choice instead of a codon that preserves the YYG andYYGXDX motifs;

(iii) Avoidance of a GGCA AID hotspot in the region of the HCDR3/FW4junction; and

(iv) Common occurrence (and thus conservation and acceptability) inhumans of the JH6*02 variant.

This rationale was tested by the inventors in laboratory examples, inorder to see if human JH6*02 could desirably participate in antibodygene segment recombination and heavy chain production in a foreign(non-human vertebrate) setting, and moreover to assess if long HCDR3sbased on human JH6*02 could be produced in vivo (in naive and immunisedsettings) in such non-human systems. It was noted that in some non-humansettings, such as a mouse, the YYG and YYGXDX motifs are not conserved,and thus the inventors decided that it was important to test whether ornot JH6*02 (having the YYG and YYGXDX motifs) could function properly insuch a foreign setting to participate in VDJ recombination and selectionagainst antigen.

Thus, as explained further in the examples, the inventors constructedtransgenic JH6*02-containing IgH loci in ES cells, generated transgenicnon-human vertebrates from the ES cells (both naive and immunised with arange of different target antigen types), isolated antibodies and heavychain sequences based on JH6*02 as well as B-cells expressing these andmade hybridomas expressing antigen-specific antibodies that are based onthe chosen JH6*02 variant. The inventors found that the JH6*02 variantwas extensively used and could contribute to the production of HCDR3 ofat least 20 amino acids in many different heavy chains (includingantigen-specific heavy chains). The chosen variant was preferably usedover other JH gene segments in all settings (naive, immunised andantigen-specific) for the production of HCDR3 of at least 20 aminoacids.

Thus, the present invention provides an IgH locus including human JH6*02(IMGT nomenclature) as a mandatory JH gene segment. In one embodiment,the locus comprises non-human vertebrate (eg, mouse or rat) constantregion gene segments downstream (ie, 3′ of) the human JH6*02; and one ormore VH gene segments (eg, a plurality of human VH gene segments) andone or more D gene segments (eg, a plurality of human D gene segments)upstream of (ie, 5′ of) the human JH6*02. For example, the locus iscomprised by a vector (eg, a DNA vector, eg, a yeast artificialchromosome (YAC), BAC or PAC). Such a vector (eg, YAC) can be introducedinto a non-human vertebrate (eg, mouse or rat) cell using standardtechniques (eg, pronuclear injection) so that the locus is integratedinto the cell genome for expression of IgH chains comprising at leastone chain whose variable domain is a product of the recombination ofhuman JH6*02 with a VH and a D gene segment.

In another example, the locus (eg, with a completely human, rat or mouseconstant region, or a human/mouse chimaeric constant region) can beprovided in the genome of a non-human vertebrate (eg, mouse or rat)cell. For example, the cell is an ES cell or an antibody-producing cell(eg, an isolated B-cell, an iPS cell or a hybridoma).

In another example, the invention provides a non-human vertebrate (eg, amouse or a rat) comprising an IgH locus of the invention which comprisesa human JH6*02 gene segment, wherein the locus can express an IgH chainwhose variable domain is a product of the recombination of human JH6*02with a VH and a D gene segment. As shown in the examples, the inventorshave successfully produced such mice which produce such IgH chains withVH domains based on human JH6*02. The inventors isolated and sequencedIgH chains from the mice before (naive) and after (immunised) exposureto a range of target antigens and confirmed by comparison to IMGT IgHgene segment sequences that the isolated chains (and antibodiescontaining these) were produced based on JH6*02. Such chains were foundin naive mice, as well as in antigen-specific antibodies from immunisedmice. B-cells were isolated from immunised mice, wherein the B-cellsexpress antibodies based on JH6*02 and hybridomas were generated fromthe B-cells, the hybridomas expressing antigen-specific antibodies basedon JH6*02. The inventors, therefore, provided the locus, vertebrate,cell and hybridoma of the invention based on the use of human JH6*02 andshowed that antibodies based on JH6*02 and B-cells expressing these canbe successfully produced and isolated following immunisation of thevertebrates, corresponding hybridomas being a good source of antibodieswhose VH domains are based on JH6*02, eg for administration to apatient, eg, for human medicine. Furthermore, it was found possible toproduce and isolated antigen-specific antibodies whose VH domains arebased on JH6*02 and which had a relatively long HCDR3 (eg, 20 aminoacids).

Thus, the present invention provides embodiments as in the followingclauses:—

1. A non-human vertebrate (optionally a mouse or a rat) or vertebratecell whose genome comprises an immunoglobulin heavy chain locuscomprising human gene segment JH6*02, one or more VH gene segments andone or more D gene segments upstream of a constant region; wherein thegene segments in the heavy chain locus are operably linked to theconstant region thereof so that the mouse is capable of producing anantibody heavy chain produced by recombination of the human JH6*02 witha D segment and a VH segment.

In another example, the invention provides

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises an immunoglobulin heavy chain locus comprisingone, more or all of human IGHV gene segments selected from V3-21, V3-13,V3-7, V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18, V4-4, V3-9, V3-23, V3-11and V3-20 (eg, one, more or all of V3-21*03, V3-13*01, V3-7*01, V6-1*01,V1-8*01, V1-2*02, V7-4-1*01, V1-3*01, V1-18*01, V4-4*01, V3-9*01 andV3-23*04). These segments were found in naive repertoires to beproductive to produce HCDR3s of at least 20 amino acids in length. In anembodiment, the locus comprises a human JH6, eg, JH6*02.

The invention also provides a HCDR3, VH domain, antibody heavy chain orantibody having a HCDR3 size of at least 20 amino acids. Optionally, theHCDR3 or VH domain (or VH domain of the heavy chain or antibody)comprises mouse AID-pattern somatic hypermutations and/or mousedTd-pattern mutations. This can be provided, for example, wherein VHdomain is produced in a mouse comprising mouse AID and/or mouse TdT (eg,endogenous AID or TdT). See also Annu. Rev. Biochem. 2007. 76:1-22;Javier M. Di Noia and Michael S. Neuberger, “Molecular Mechanisms ofAntibody Somatic Hypermutation” (in particular FIG. 1 and associateddiscussion on AID hotspots in mouse); and Curr Opin Immunol. 1995 April;7(2):248-54, “Somatic hypermutation”, Neuberger M S and Milstein C (inparticular, discussion on hotspots in mouse), the disclosures of whichare incorporated herein by reference.

These segments were found in naive repertoires to be productive inrecombination with human JH6*02 to produce HCDR3s of at least 20 aminoacids in length.

In an example, the vertebrate is naive. In another embodiment, thevertebrate instead is immunised with a target antigen.

In an example, the vertebrate or cell mentioned below is capable of soproducing an antibody heavy chain upon immunisation with a targetantigen. In an example, the vertebrate is an immunised vertebrate thatproduces antibody heavy chains specific for a target antigen and whereinthe variable domains of the heavy chains are the product ofrecombination between a VH, D and JH6*02. For example, the D is selectedfrom human D3-3, D2-15, D3-9; D4-17; D3-10; D2-2; D5-24; D6-19; D3-22;D6-13; D5-12; D1-26; D1-20; D5-18; D3-16; D2-21; D1-14; D7-27; D1-1;D6-25; D2-14 and D4-23 (eg, selected from D3-9*01; D4-17*01; D3-10*01;D2-2*02; D5-24*01; D6-19*01; D3-22*01; D6-13*01; D5-12*01; D1-26*01;D1-20*01; D5-18*01; D3-16*02; D2-21*02; D1-14*01; D7-27*02; D1-1*01;D6-25*01; D2-15*01; and D4-23*01). For example, the D is human D3-9 orD3-10. In an example, the HCDR3 length is at least 20 amino acids (eg,20, 21, 23 or 24).

In an example of the vertebrate or cell, the genome comprises additionalhuman JH gene segments (eg, JH2, 3, 4 and 5 gene segments).

In an example of the vertebrate or cell, the genome comprises animmunoglobulin light chain locus comprising one or more human V genesegments and one or more human J gene segments upstream of a constantregion (eg, a human or a mouse lambda or kappa constant region).

For rearrangement and expression of heavy chains, the locus comprisescontrol elements, such as an Ê and Ŝ between the J gene segment(s) andthe constant region as is known by the skilled person. In one example, amouse Ê and Ŝ is included in the heavy chain locus between the JH6*02and the constant region (ie, in 5′ to 3′ order the locus comprises theJH6*02, Ê and Ŝ and constant region). In an example, the Ê and Ŝ are Êand Ŝ of a mouse 129-derived genome (eg, a 129Sv-derived genome, eg,129Sv/EV (such as 129S7Sv/Ev (such as from AB2.1 or AB2.2 cellsobtainable from Baylor College of Medicine, Texas, USA) or129S6Sv/Ev))); in another example, the Ê and Ŝ are Ê and Ŝ of a mouseC57BL/6-derived genome. In this respect, the locus can be constructed inthe IgH locus of the genome of a cell selected from AB2.1, AB2.2, VGF1,CJ7 and FH14. VGF1 cells were established and described in Auerbach W,Dunmore J H, Fairchild-Huntress V, et al; Establishment and chimeraanalysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem celllines. Biotechniques 2000; 29:1024-8, 30, 32, incorporated herein byreference.

Additionally or alternatively, the constant region (or at least a Ĉ or Ĉand gamma constant regions thereof) is a constant region (or Ĉ or Ĉ andgamma constant regions thereof) is of a genome described in theparagraph immediately above.

A suitable source of JH6*02 and other human DNA sequences will bereadily apparent to the skilled person. For example, it is possible tocollect a DNA sample from a consenting human donor (eg, a cheek swabsample as per the Example herein) from which can be obtained suitableDNA sequences for use in constructing a locus of the invention. Othersources of human DNA are commercially available, as will be known to theskilled person. Alternatively, the skilled person is able to constructgene segment sequence by referring to one or more databases of human Iggene segment sequences disclosed herein.

2. The vertebrate of clause 1, wherein the vertebrate has been immunisedwith a target antigen and wherein the variable domain of the heavy chainis the product of recombination between a VH, D and JH6*02 and whereinthe HCDR3 length is at least 20 amino acids (eg, 20, 21, 23 or 24).

Optionally, the immunised vertebrate produces an antibody heavy chainspecific for a target antigen and wherein the variable domain of theheavy chain is the product of recombination between a VH, D and JH6*02and wherein the HCDR3 length is at least 20 amino acids (eg, 20, 21, 23or 24).

3. A non-human vertebrate cell (optionally a mouse cell or a rat cell)whose genome comprises an immunoglobulin heavy chain locus comprisinghuman gene segment JH6*02, one or more VH gene segments and one or moreD gene segments upstream of a constant region; wherein the gene segmentsin the heavy chain locus are operably linked to the constant regionthereof for producing (eg, in a subsequent progeny cell) an antibodyheavy chain produced by recombination of the human JH6*02 with a Dsegment and a VH segment.

4. The cell of clause 3, which is an ES cell capable of differentiationinto a progeny antibody-producing cell that expresses said heavy chain.

5. The vertebrate or cell of any preceding clause, wherein the heavychain locus comprises a human JH6*02 recombination signal sequence (RSS)operably connected 5′ to the JH6*02 gene segment.

For example, the native RSS-JH6*02 sequence can be used toadvantageously maintain the natural pairing between RSS and this JH genesegment. In this respect, the following sequence is used:—

ggtttttgtggggtgaggatggacattctgccattgtgattactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcag (SEQ ID NO: 238)

RSSs have a common architecture: 9mer (eg, first underlined sequenceabove) followed by a 22 bp spacer and then a 7mer (eg, second underlinedsequence above). Spacers are 23 bp+/−1 normally, while the 9 and 7merare more conserved.

6. The vertebrate or cell of clause 5, wherein the RSS is SEQ ID NO: 238or a sequence having an identical 9mer and 7mer sequence flanking asequence that is at least 70% identical to the 22mer sequence of SEQ IDNO: 238.

7. The vertebrate or cell of clause 6, wherein the RSS and JH6*02 areprovided as SEQ ID NO: 237.

8. The vertebrate or cell of any preceding clause, wherein the JH6*02 isthe only JH6-type gene segment in the genome.

9. The vertebrate or cell of any preceding clause, wherein the JH6*02 isthe closest JH gene segment to the constant region in the locus.

10. The vertebrate or cell of any preceding clause, wherein the locuscomprises one, more or all human D gene segments D3-9; D4-17; D3-10;D2-2; D5-24; D6-19; D3-22; D6-13; D5-12; D1-26; D1-20; D5-18; D3-16;D2-21; D1-14; D7-27; D1-1; D6-25; D2-14; and D4-23.

For example, the locus comprises one, more or all of human D genesegments D3-9*01; D4-17*01; D3-10*01; D2-2*02; D5-24*01; D6-19*01;D3-22*01; D6-13*01; D5-12*01; D1-26*01; D1-20*01; D5-18*01; D3-16*02;D2-21*02; D1-14*01; D7-27*02; D1-1*01; D6-25*01; D2-15*01; and D4-23*01.

11. The vertebrate or cell of clause 10, wherein the locus comprisesone, more or all human D gene segments D3-9, D3-10, D6-19, D4-17, D6-13,D3-22, D2-2, D2-25 and D3-3.

These D segments were found to be productive in recombination with humanJH6*02 to produce HCDR3s of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human D genesegments D3-9, D3-10, D6-19, D4-17, D6-13 and D3-22 (for example one,more or all of D3-9*01, D3-10*01, D6-19*01, D4-17*01, D6-13*01 andD3-22*01). These D segments were found in naive repertoires to beproductive in recombination with human JH6*02 to produce HCDR3s of atleast 20 amino acids in length.

In an example, the locus comprises one, more or all human D genesegments D3-10, D6-19 and D1-26 (for example, one, more or all ofD3-10*01, D6-19*01 and D1-26*01). These D segments were found inimmunised repertoires to be productive in recombination with humanJH6*02 to produce HCDR3s of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human D genesegments D3-9 and D3-10 (for example, one, more or all of D3-9*01 andD3-10*01). These D segments were found in antigen-specific repertoiresto be productive in recombination with human JH6*02 to produce HCDR3s ofat least 20 amino acids in length.

12. The vertebrate or cell of any preceding clause, wherein the locuscomprises a plurality of human D gene segments and the JH6*02 is inhuman germline configuration with respect to the 3′-most human D genesegment (or all of the human D segments comprised by the locus).

In an example, the 3′-most D gene segment is D7-27. In an example, thelocus comprises all of human D gene segments from D1-1 to D7-27 aspresent in a germline human IgH locus (eg, as shown in the IMGTdatabase).

Alternatively or additionally, the JH6*02 is in human germlineconfiguration with respect to one, more or all of the Ê Ŝ and constantregion (eg, Cu) 13. The vertebrate or cell of any preceding clause,wherein the locus comprises one, more or all of IGHV gene segmentsselected from V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18,V4-4, V3-9, V3-23, V3-11 and V3-20.

In an example, the locus comprises one, more or all human IGHV genesegments V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18,V4-4, V3-9, V3-23 (for example, one, more or all of V3-21*03, V3-13*01,V3-7*01, V6-1*01, V1-8*01, V1-2*02, V7-4-1*01, V1-3*01, V1-18*01,V4-4*01, V3-9*01 and V3-23*04). These segments were found in naiverepertoires to be productive in recombination with human JH6*02 toproduce HCDR3s of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human IGHV genesegments V3-7, V3-11 and V4-4 (for example, one, more or all of V3-7*01,V3-11*01 and V4-4*02). These segments were found in immunisedrepertoires to be productive in recombination with human JH6*02 toproduce HCDR3s of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human IGHV genesegments V4-4, V1-8, V3-9, V3-11 and V3-20 (for example, one, more orall of V4-4*02, V1-8*01, V3-9*01, V3-11*01 and V3-20 (eg, *d01). Thesesegments were found in antigen-specific repertoires to be productive inrecombination with human JH6*02 to produce HCDR3s of at least 20 aminoacids in length.

14. The vertebrate or cell of any preceding clause, wherein the locuscomprises one, more or all of human D3-9*01, D3-10*01, D6-19*01,D6-13*01, D1-26*01, IGHV1-8*01, IGHV4-61*01, IGHV6-1*01, IGHV4-4*02,IGHV1-3*01, IGHV3-66*03, IGHV3-7*01 and IGHV3-9*01.

These are gene segments that very frequently combine with JH6*02 toproduce productive heavy chains and antibodies.

For example, the locus comprises one, more or all of human IGHV1-8*01,D3-9*01 and D3-10*01. These gene segments were productive with JH6*02 toproduce HCDR3s of at least 20 amino acids in more than 10 antibodies.

15. An antibody-producing cell (eg, a B-cell) that is a progeny of thecell of any one of clauses 3 to 14, wherein the antibody-producing cellcomprises a heavy chain locus comprising a rearranged variable regionproduced by recombination of human JH6*02 with a D segment and a VHsegment (eg, JH6*02 with human VH3-11 (eg, VH3-11*01) and D3-9; VH3-20(eg, VH3-20*01) and D3-10; VH4-4 (eg, VH4-4*02) and D3-10; VH3-9 (eg,VH3-9*01) and D3-10; or VH1-8 (eg, VH1-8*01) and D310).

Such a variable region would be the product of in vivo somatichypermutation in a non-human vertebrate or cell of the invention.

16. The cell of clause 15, which is a B-cell or hybridoma that expressesa target antigen-specific antibody comprising a heavy chain thatcomprises a rearranged variable region produced by recombination ofhuman JH6*02 with a D segment and a VH segment (eg, JH6*02 with humanVH3-11 (eg, VH3-11*01) and D3-9; VH3-20 (eg, VH3-20*01) and D3-10; VH4-4(eg, VH4-4*02) and D3-10; VH3-9 (eg, VH3-9*01) and D3-10; or VH1-8 (eg,VH1-8*01) and D310).

Such a variable region would be the product of in vivo somatichypermutation in a non-human vertebrate or cell of the invention

17. The vertebrate or cell of any preceding clause, wherein the antibodyheavy chain specifically binds a target antigen.

18. The vertebrate or cell of any preceding clause, wherein the antibodyheavy chain has a HCDR3 length of at least 20 amino acids.

Optionally, the HCDR3 length is at least 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 amino acids. Additionally, in one example the length is no morethan 35, 34, 33, 32 or 31 amino acids. For example, the HCDR3 length is20, 21, 22, 23 or 24 amino acids.

19. The vertebrate or cell of any preceding clause, wherein the antibodyheavy chain is a product of the recombination of JH6*02 with a human VHgene segment recited in clause 13 or 14 and/or a D gene segment recitedin clause 10, 11 or 14.

20. The vertebrate or cell of any preceding clause, wherein allendogenous non-human vertebrate heavy chain variable region genesegments have been inactivated in the genome (Eg, by gene segmentdeletion or inversion).

21. The vertebrate or cell of any preceding clause, wherein the genomeis homozygous for said heavy chain locus.

22. A heavy chain (eg, comprised by an antibody) isolated from avertebrate of any one of clauses 1, 2, 5 to 14 and 17 to 21 wherein theheavy chain comprises a HCDR3 of at least 20 amino acids.

23. The heavy chain of clause 22, wherein the HCDR3 is the product ofrecombination of human JH6*02 with a human VH gene segment recited inclause 13 or 14 and/or a D gene segment recited in clause 10, 11 or 14.

In an example, the heavy chain is chimaeric where the C region isnon-human. In an example, the heavy chain is human where the C region ishuman.

24. A heavy chain (eg, comprised by an antibody) whose VH variabledomain is identical to the VH variable domain of the heavy chain ofclause 22 or 23, and which comprises a human constant region or ahuman-mouse chimaeric constant region (eg, CH1 is human and the otherconstant domains are mouse).

25. The heavy chain of clause 22, 23 or 24, whose VH variable domain isspecific for a target antigen.

26. A method for producing a heavy chain, VH domain or an antibodyspecific to a target antigen, the method comprising immunizing anon-human vertebrate according to any one of clauses 1, 2, 5 to 14 and17 to 21 with the antigen and isolating the heavy chain, VH domain or anantibody specific to a target antigen or a cell producing the heavychain, VH domain or an antibody, wherein the heavy chain, VH domain oran antibody comprises a HCDR3 that is derived from the recombination ofhuman JH6*02 with a VH gene segment and a D gene segment.

27. A method for producing a human heavy chain or antibody comprisingcarrying out the method of clause 26, wherein the constant region of thelocus is a non-human vertebrate (eg, mouse or rat) constant region, andthen replacing the non-human constant region of the isolated heavy chainor antibody with a human constant region (eg, by engineering of thenucleic acid encoding the antibody).

28. A heavy chain, VH domain or an antibody produced by the method ofclause 26 or 27. Optionally the HCDR3 length is at least 20 amino acidsas herein described.

29. A B-cell or hybridoma expressing a heavy chain VH domain that isidentical to the VH domain of the heavy chain of clause 22, 23 or 28.

30. A nucleic acid encoding the VH domain of the heavy chain of clause22, 23 or 28, or encoding the heavy chain of clause 22, 23, 24, 25 or28.

31. A vector (eg, a CHO cell or HEK293 cell vector) comprising thenucleic acid of clause 30; optionally wherein the vector is in a hostcell (eg, a CHO cell or HEK293 cell).

32. A pharmaceutical composition comprising the antibody, heavy chain orVH domain (eg, comprised by an antibody) of any one of clauses 22 to 25and 28, together with a pharmaceutically-acceptable excipient, diluentor a medicament (eg, a further antigen-specific variable domain, heavychain or antibody).

33. The antibody, heavy chain or VH domain (eg, comprised by anantibody) of any one of clauses 22 to 25 and 28 for use in medicine (eg,human medicine).

For example, the locus comprises the following human VH gene segments

IGHV6-1

IGHV3-7

IGHV1-8

IGHV3-9

IGHV3-11

IGHV3-13

IGHV1-18

IGHV3-30

IGHV4-31

IGHV4-39 IGHV4-59

Optionally also (i) and/or (ii)

(i)

IGHV1-2 IGHV2-5 and IGHV3-21

(ii)

IGHV1-2 IGHV2-5 IGHV3-21 IGHV1-24

For example, the locus comprises the following human VH gene segmentvariants

IGHV6-1*01

IGHV3-7*01

IGHV1-8*01

IGHV3-9*01

IGHV3-11*01

IGHV3-13*01

IGHV1-18*01

IGHV3-30*18

IGHV4-31*03

IGHV4-39*01 and

IGHV4-59*01;

Optionally also (iii) or (iv)

(ii)

IGHV1-2*04 IGHV2-5*10 and IGHV3-21*03

(iv)

IGHV1-2*02 IGHV2-5*01 IGHV3-21*01 and IGHV1-24*01

For example, the locus comprises the following human JH gene segmentvariants

IGHJ2*01 IGHJ3*02

IGHJ4*02 IGHJ5*02 and IGHJ6*02

For example, the locus comprises the following human D gene segments

IGHD1-1

IGHD2-2

IGHD3-9

IGHD3-10

IGHD5-12

IGHD6-13

IGHD1-14

IGHD2-15

IGHD3-16

IGHD4-17

IGHD6-19

IGHD2-21

IGHD5-24

IGHD1-26 and

IGHD7-27

and optionally also (v) or (vi)

(v)

IGHD3-3

(vi)

IGHD3-3

IGHD4-4

IGHD5-5

IGHD6-6

IGHD1-7

IGHD2-8 and

IGHD2-8

The Present Invention Provides in a Fifth Configuration—

Constant Regions Tailored to Human Use & Antibody Humanisation

Additional rational design and bioinformatics has led the inventors torealise that specific human constant region variants are conservedacross many diverse human populations. The inventors realised that thisopens up the possibility of making a choice to humanise antibodies,chains and variable domains by using such specific constant regions inproducts, rather than arbitrarily choosing the human constant region (ora synthetic version of a human constant region). This aspect of theinvention also enables one to tailor antibody-based drugs to specifichuman ethnic populations, thereby more closely matching drug to patient(and thus disease setting) than has hitherto been performed. It can be aproblem in the state of the art that antibodies are humanised with anarbitrary choice of human constant region (presumably derived from one(often unknown) ethnic population or non-naturally occurring) that doesnot function as well in patients of a different human ethnic population.This is important, since the constant region has the major role inproviding antibody effector functions, eg, for antibody recycling,cellular and complement recruitment and for cell killing.

As discussed further in WO2011066501, human IgG sub-types IgG1, IgG2,gG3 and IgG4 exhibit differential capacity to recruit immune functions,such as antibody-dependent cellular cytotoxicity (ADCC, e.g., IgG1 andIgG3), antibody-dependent cellular phagocytosis (ADCP, e.g., IgG1, IgG2,IgG3 and IgG4), and complement dependent cytotoxicity (CDC, e.g., IgG1,IgG3). Sub-type-specific engagement of such immune functions is based onselectivity for Fc receptors on distinct immune cells and the ability tobind C1q and activate the assembly of a membrane attack complex (MAC).

Among the various types, relative affinity for FcY receptors (e.g.,FcYRI, FcYRIIa/b/c, FcYRIIIa/b) is high for IgG1 and IgG3, however,there is minimal affinity for IgG2 (restricted to the FcYRIIa 131Hpolymorphism), and IgG4 only has measurable affinity for FcYRI. Usingcomparative sequence analysis and co-crystal structures, the key contactresidues for receptor binding have been mapped to the amino acidresidues spanning the lower hinge and CH2 region. Using standard proteinengineering techniques, some success in enhancing or reducing theaffinity of an antibody preparation for Fc receptors and the C1qcomponent of complement has been achieved.

Among the isotypes, IgG2 is least capable of binding the family of Fcreceptors. Using IgG2 as the starting point, efforts have been made tofind a mutant with diminished effector functions but which retains FcRnbinding, prolonged stability, and low immunogenicity. Improved mutantsof this nature may provide improved antibody therapeutics with retainedsafety. Human IgG1 therapeutic antibodies that bind to cell surfacetargets are able to engage effector cells that may mediate cell lysis ofthe target cell by antibody-dependent cellular cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC). These mechanisms occur throughinteraction of the CH2 region of the antibody Fc domain to FcyRreceptors on immune effector cells or with C1q, the first component ofthe complement cascade. Table 19 shows the activities of different humangamma sub-types. The skilled person may choose accordingly to promote ordampen-down activity depending upon the disease setting in humans ofinterest. For example, use of a human gamma-1 constant region isdesirable when one wishes to isolated totally human heavy chains andantibodies that have relatively high complement activation activity bythe classical pathway and FcYR1 recognition in human patients. See alsoMol Immunol. 2003 December; 40(9):585-93; “Differential binding to humanFcgamma RIIa and Fcgamma RIIb receptors by human IgG wild type andmutant antibodies”; Armour K L et al, which is incorporated herein byreference.

IgG2 constant regions are well suited to producing antibodies and heavychains according to the invention for binding to cytokines or solubletargets in humans, since IgG2 is essentially FcγRI,III-silent,FcYRIIa-active and has little Complement activity.

IgG1 constant regions have wide utility for human therapeutics, sinceIgG1 antibodies and heavy chains are FcγRI,II,III-active and havecomplement activity. This can be enhanced by using a human gamma-1constant region that has been activated by engineering as is known inthe art.

The work of the inventors has therefore identified a collection of humanconstant region of different isotypes from which an informed choice canbe made when humanising chimaeric antibody chains (or conjugating Vdomains, such as dAbs or Camelid VHH, to constant regions). Thecollection was identified on the basis of bioinformatics analysis of the1000 Genomes database, the inventors selecting constant region variantsthat are frequently occurring across several human ethnic populations,as well as those that appear with relatively high frequency withinindividual populations (as assessed by the number of individuals whosegenomes comprise the variant). By sorting through the myriad possiblesequences on this basis, the inventors have provided a collection ofhuman constant region variants that are naturally-occurring and whichcan be used when rationally designing

antibodies, heavy chains and other antibody-based formats that bear ahuman constant region. In particular, this is useful when humanisingchimaeric heavy chains to produce totally human chains in which both thevariable and constant regions are human. This is useful forcompatibility with human patients receiving antibody-based drugs.

To this End, the Invention Provides the Following Aspects:—

1. method of producing an antibody heavy chain, the method comprising

(a) providing an antigen-specific heavy chain variable domain (eg, VH(such as a human VH or dAb) or VHH or a humanised heavy chain variabledomain); and

(b) combining the variable domain with a human heavy chain constantregion to produce an antibody heavy chain comprising (in N- toC-terminal direction) the variable domain and the constant region;

wherein

the human heavy chain constant region is an IGHAref, IGHA1a, IGHA2a,IGHA2b, IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref,IGHG4a, IGHDref, IGHEref, IGHMref, IGHMa or IGHMb constant region.

Step (b) can be carried out, eg, using recombinant DNA technology usingthe corresponding nucleotide sequences.

For the constant region according to any aspect of this configuration,either genomic DNA or equivalent (ie, having introns and exons andoptionally also 5′ UTR sequences, eg, with native or a non-native leadersequence) can be used for the constant region. For example, any of the“GENOMIC” sequences disclosed as SEQ ID NO: 365 onwards herein.Alternatively, an intronless sequence can be used, for example any ofthe “CDS” sequences disclosed as SEQ ID NO: 365 onwards herein (eg, withnative or a non-native leader sequence).

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHAref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHA1a constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHA2a constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHA2b constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is IGHG1 ref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHG2ref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHG2a constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHG3ref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHG3a constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHG3b constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHG4ref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHG4a constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHDref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHEref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHMref constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHMa constant region.

Optionally for any aspect of this configuration of the invention, thehuman heavy chain constant region is an IGHMb constant region.

Optionally, a derivative (eg, a mutant or conjugate) of the heavy chainor an antibody containing the heavy chain is produced. For example, atoxic payload can be conjugated (eg, for oncology applications). Forexample, one or more mutations can be introduced, as is known in theart, to inactivate or enhance Fc effector function.

2. The method of aspect 1, wherein the variable domain is a humanvariable domain.

A human variable domain is, for example, the product of recombination ina transgenic non-human vertebrate of human VH, D and JH gene segments.Alternatively, the variable domain is identified using in vitro displaytechnology from a human VH library, eg, using phage display, ribosomedisplay or yeast display, as is known in the art.

In another embodiment, the variable domain is a humanised variabledomain, eg, comprising human frameworks with non-human (eg, mouse orrat) CDRs). Humanisation technology is conventional in the art, and willbe readily known to the skilled person.

3. The method of any preceding aspect, wherein the variable domain haspreviously been selected from a non-human vertebrate that has beenimmunised with the antigen.

For example, the vertebrate (such as a mouse or rat) genome comprises achimaeric heavy chain locus comprising a human variable region (human V,D and JH gene segments) operably connected upstream of a non-humanvertebrate constant region so that the locus is able to rearrange forthe expression of heavy chains comprising human variable domains andnon-human vertebrate constant regions.

In alternative embodiments, the variable domain is selected using an invitro technology such as phage display, ribosome display or yeastdisplay. In this case the variable domain may be displayed with orwithout a constant region, provided that it is later combined with ahuman constant region as per the invention.

4. The method of any preceding aspect, comprising providing anexpression vector (Eg, a mammalian expression vector, such as a CHO orHEK293 vector) comprising a nucleotide sequence encoding the constantregion; inserting a nucleotide sequence encoding the variable domaininto the vector 5′ of the constant region sequence; inserting the vectorinto a host cell and expressing the heavy chain by the host cell; themethod further comprising isolating a heavy chain (eg, as part of anantibody) comprising the variable domain and the human constant region.

The vector comprises regulatory elements sufficient to effect expressionof the heavy chain when the vector is harboured by a host cell, eg, aCHO or HEK293 cell.

5. The method of any preceding aspect, further comprising obtaining anucleotide sequence encoding the heavy chain.

6. An antibody comprising a human heavy chain, the heavy chaincomprising a variable domain that is specific for an antigen and aconstant region that is an IGHAref, IGHA1a, IGHA2a, IGHA2b, IGHG1ref,IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref, IGHG4a, IGHDref,IGHEref, IGHMref, IGHMa or IGHMb constant region.

7. A polypeptide comprising (in N- to C-terminal direction) a leadersequence, a human variable domain that is specific for an antigen and ahuman constant region that is an IGHAref, IGHA1a, IGHA2a, IGHA2b,IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref, IGHG4a,IGHDref, IGHEref, IGHMref, IGHMa or IGHMb constant region; wherein (i)the leader sequence is not the native human variable domain leadersequence (eg, the leader sequence is another human leader sequence or anon-human leader sequence); and/or (ii) the variable domain comprisesmouse AID-pattern somatic mutations or mouse terminal deoxynucleotidyltransferase (TdT)-pattern junctional mutations.

8. A nucleotide sequence encoding (in 5′ to 3′ direction) a leadersequence and a human antibody heavy chain, the heavy chain comprising avariable domain that is specific for an antigen and a constant regionthat is an IGHAref, IGHA1a, IGHA2a, IGHA2b, IGHG1ref, IGHG2ref, IGHG2a,IGHG3ref, IGHG3a, IGHG3b, IGHG4ref, IGHG4a, IGHDref, IGHEref, IGHMref,IGHMa or IGHMb constant region; and the leader sequence being operablefor expression (eg, in a mammalian CHO or HEK293 cell) of the heavychain and wherein the leader sequence is not the native human variabledomain leader sequence (eg, the leader sequence is another human leadersequence or a non-human leader sequence).

In an example, the leader sequence is

ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGGCGT GCACAGC

Which translates to

MGWSCIILFLVATATGVHS

9. A nucleotide sequence encoding (in 5′ to 3′ direction) a promoter anda human antibody heavy chain, the heavy chain comprising a variabledomain that is specific for an antigen and a constant region that is anIGHAref, IGHA1a, IGHA2a, IGHA2b, IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref,IGHG3a, IGHG3b, IGHG4ref, IGHG4a, IGHDref, IGHEref, IGHMref, IGHMa orIGHMb constant region; and the promoter being operable for expression(eg, in a mammalian CHO or HEK293 cell) of the heavy chain and whereinthe promoter is not the native human promoter.

In one embodiment, the promoter sequence is a human IGK 3-15 promoter.

10. The antibody, polypeptide or nucleotide sequence of any one ofaspects 6 to 9, wherein the variable domain comprises mouse AID-patternsomatic mutations and/or mouse terminal deoxynucleotidyl transferase(TdT)-pattern junctional mutations.

For example, one way, in any aspect of this configuration of theinvention, to provide mouse AID-pattern somatic mutations and/or mouseterminal deoxynucleotidyl transferase (TdT)-pattern junctional mutationsis to select a variable domain from a non-human vertebrate or cell. Forexample, a vertebrate or cell as disclosed herein.

11. A vector (eg, a CHO cell or HEK293 cell vector) comprising thenucleic acid of aspect 8, 9 or 10; optionally wherein the vector is in ahost cell (eg, a CHO cell or HEK293 cell).

12. A pharmaceutical composition comprising the antibody or polypeptideof any one of aspects 6, 7 and 10, together with apharmaceutically-acceptable excipient, diluent or a medicament (eg, afurther antigen-specific variable domain, antibody chain or antibody).

13. The antibody or polypeptide of any one of aspects 6, 7 and 10 foruse in treating and/or preventing a medical condition in a humanpatient.

14. Use of the antibody or polypeptide of any one of aspects 6, 7 and 10for the manufacture of a medicament for treating and/or preventing amedical condition in a human patient.

15. The antibody, polypeptide or use of aspect 13 or 14, wherein thehuman is a member of a human population selected from population numbers1-14, wherein the populations are numbered as follows (population labelsbeing according to 1000 Genomes Project nomenclature)

1=ASW;

2=CEU;

3=CHB;

4=CHS;

5=CLM;

6=FIN;

7=GBR;

8=IBS;

9=JPT;

10=LWK;

11=MXL;

12=PUR;

13=TSI;

14=YRI.

16. The antibody, polypeptide or use of aspect 15, wherein the constantregion is a

(i) IGHA1a constant region and the human population is selected from anypopulation number 1-14;

(ii) IGHA2a constant region and the human population is selected fromany population number 1-14;

(iii) IGHA2b constant region and the human population is selected fromany population number 1-14;

(iv) IGHG2a constant region and the human population is selected fromany population number 1-9 and 11-13;

(v) IGHG3a constant region and the human population is selected from anypopulation number 1-14;

(vi) IGHG3b constant region and the human population is selected fromany population number 1-8 and 11-13;

(vii) IGHG4a constant region and the human population is selected fromany population number 1-9 and 11-13;

(viii) IGHMa constant region and the human population is selected fromany population number 1-14; or

(ix) IGHMb constant region and the human population is selected from anypopulation number 1-14;

Wherein the populations are numbered as follows (population labels beingaccording to 1000 Genomes Project nomenclature)

1=ASW;

2=CEU;

3=CHB;

4=CHS;

5=CLM;

6=FIN;

7=GBR;

8=IBS;

9=JPT;

10=LWK;

11=MXL;

12=PUR;

13=TSI;

14=YRI.

17. A vector (eg, a CHO cell or HEK293 cell vector) comprising aIGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4aconstant region nucleotide sequence that is 3′ of a cloning site for theinsertion of a human antibody heavy chain variable domain nucleotidesequence, such that upon insertion of such a variable domain sequencethe vector comprises (in 5′ to 3′ direction) a promoter, a leadersequence, the variable domain sequence and the constant region sequenceso that the vector is capable of expressing a human antibody heavy chainwhen present in a host cell.

The Present Invention Provides in a Sixth Configuration—

Multiple Variants in the Same Genome Cis or Trans

The inventors' analysis has revealed groupings of naturally-occurringhuman antibody gene segment variants as set out in Table 13 and Table14. This revealed the possibility of producing transgenic genomes innon-human vertebrates and cells wherein the genomes contain more thanthe natural human complement of specific human gene segments. In oneexample, this can be achieved by providing more than the natural humancomplement of a specific gene segment type on one or both of therespective Ig locus (eg, one or both chromosomes harbouring IgH in amouse genome or mouse cell genome).

To this End, this Configuration of the Invention Provides the Following(as Set Out in Numbered Paragraphs):—

1. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 3human variable region gene segments of the same type (eg, at least 3human VH6-1 gene segments, at least 3 human JH6 gene segments, at least3 human VK1-39 gene segments, at least 3 human D2-2 gene segments or atleast 3 human JK1 gene segments), wherein at least two of the human genesegments are variants that are not identical to each other.

For example, the genome comprises a variable region that comprises V, Dand J gene segments (for the variable region of a heavy chain locus) orV and J gene segments (for the variable region of a light chain locus)upstream of a constant region for expression of heavy or light chainsrespectively.

In an alternative, the skilled person can choose to provide more thanthe wild type human complement of a specific gene segment type byproviding several copies of one variant type of the human gene segment.Thus, there is provided A non-human vertebrate (eg, a mouse or rat) or anon-human vertebrate cell (eg, an ES cell or a B-cell) having a genomecomprising at least 3 human variable region gene segments of the sametype (eg, at least 3 human VH6-1 gene segments, at least 3 human JH6gene segments, at least 3 human VK1-39 gene segments, at least 3 humanD2-2 gene segments or at least 3 human JK1 gene segments), wherein thehuman gene segments are identical variants.

For example, the genome comprises a variable region that comprises V, Dand J gene segments (for the variable region of a heavy chain locus) orV and J gene segments (for the variable region of a light chain locus)upstream of a constant region for expression of heavy or light chainsrespectively.

2. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different non-endogenous variable region gene segments of the same type(eg, at least 2 human VH6-1 gene segments, at least 3 human JH6 genesegments, at least 2 human VK1-39 gene segments, at least 2 human D2-2gene segments or at least 2 human JK1 gene segments) cis at the same Iglocus.

In an alternative, the skilled person can choose to provide more thanthe wild type human complement of a specific gene segment type byproviding several copies of one variant type of the human gene segment.Thus, there is provided

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2non-endogenous variable region gene segments of the same variant type(eg, at least 2 human JH6*02 gene segments) cis at the same Ig locus.

3. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different human variable region gene segments of the same type (eg, atleast 2 human VH6-1 gene segments, at least 2 human JH6 gene segments,at least 2 human VK1-39 gene segments, at least 2 human D2-2 genesegments or at least 2 human JK1 gene segments) trans at the same Iglocus; and optionally a third human gene segment of the same type,wherein the third gene segment is cis with one of said 2 different genesegments.

In an alternative, the skilled person can choose to provide more thanthe wild type human complement of a specific gene segment type byproviding several copies of one variant type of the human gene segment.Thus, there is provided A non-human vertebrate (eg, a mouse or rat) or anon-human vertebrate cell (eg, an ES cell or a B-cell) having a genomecomprising at least 2 different human variable region gene segments ofthe same variant type (eg, at least 2 human JH6*02 gene segments) transat the same Ig locus; and optionally a third human gene segment of thesame variant type, wherein the third gene segment is cis with one ofsaid 2 different gene segments.

4. A population of non-human vertebrates (eg, mice or rats) comprising arepertoire of human variable region gene segments, wherein the pluralitycomprises at least 2 human variable region gene segments of the sametype (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6gene segments, at least 2 human VK1-39 gene segments, at least 2 humanD2-2 gene segments or at least 2 human JK1 gene segments), a first ofsaid different gene segments is provided in the genome of a firstvertebrate of the population, and a second of said different genesegments being provided in the genome of a second vertebrate of thepopulation, wherein the genome of the first vertebrate does not comprisethe second gene segment.

5. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different non-endogenous variable region gene segments of the same type(eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 genesegments, at least 2 human VK1-39 gene segments, at least 2 human D2-2gene segments or at least 2 human JK1 gene segments), wherein the genesegments are derived from the genome sequence of different humanindividuals that are not genetically related over at least 3generations.

6. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 3 humanvariable region gene segments of the same type (eg, at least 3 humanVH6-1 gene segments, at least 3 human JH6 gene segments, at least 3human VK1-39 gene segments, at least 3 human D2-2 gene segments or atleast 3 human JK1 gene segments), wherein at least two of the human genesegments are variants that are not identical to each other.

7. A method of enhancing the immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous variable region gene segments of the same type (eg, atleast 2 human VH6-1 gene segments, at least 2 human JH6 gene segments,at least 2 human VK1-39 gene segments, at least 2 human D2-2 genesegments or at least 2 human JK1 gene segments) cis at the same Iglocus.

8. A method of enhancing the immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differenthuman variable region gene segments of the same type (eg, at least 2human VH6-1 gene segments, at least 2 human JH6 gene segments, at least2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or atleast 2 human JK1 gene segments) trans at the same Ig locus; andoptionally a third human gene segment of the same type, wherein thethird gene segment is cis with one of said 2 different gene segments.

9. A method of providing an enhanced human immunoglobulin variableregion gene segment repertoire, the method comprising providing apopulation of non-human vertebrates (eg, a mouse or rat) comprising arepertoire of human variable region gene segments, wherein the methodcomprises providing at least 2 different human variable region genesegments of the same type (eg, at least 2 human VH6-1 gene segments, atleast 2 human JH6 gene segments, at least 2 human VK1-39 gene segments,at least 2 human D2-2 gene segments or at least 2 human JK1 genesegments), wherein a first of said different gene segments is providedin the genome of a first vertebrate of the population, and a second ofsaid different gene segments is provided in the genome of a secondvertebrate of the population, wherein the genome of the first vertebratedoes not comprise the second gene segment.

10. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous variable region gene segments of the same type (eg, atleast 2 human VH6-1 gene segments, at least 2 human JH6 gene segments,at least 2 human VK1-39 gene segments, at least 2 human D2-2 genesegments or at least 2 human JK1 gene segments), wherein the genesegments are derived from the genome sequence of different humanindividuals that are not genetically related over at least 3generations.

11. The vertebrate, cell or method of any preceding paragraph, whereinat least 2 or 3 of said different gene segments are provided cis at thesame Ig locus in said genome.

12. The vertebrate, cell or method of any preceding paragraph, whereinthe gene segments are derived from the genome sequence of differenthuman individuals that are not genetically related over at least 3generations.

13. The vertebrate, cell or method of any preceding paragraph, whereinthe gene segments are derived from the genome sequence of two or moredifferent human individuals; optionally wherein the different humanindividuals are from different human populations.

14. The vertebrate, cell or method of paragraph 13, wherein theindividuals are not genetically related.

15. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 humanvariable region gene segments of the same type (eg, at least 2 humanVH6-1 gene segments, at least 2 human JH6 gene segments, at least 2human VK1-39 gene segments, at least 2 human D2-2 gene segments or atleast 2 human JK1 gene segments), wherein the gene segments are derivedfrom the genome sequence of different human individuals that are notgenetically related over at least 3 generations; optionally wherein atleast 2 or 3 of said different gene segments are provided at the same Iglocus in said genome.

16. The method of paragraph 15, wherein the different human individualsare from different human populations.

17. The method of paragraph 15, wherein the individuals are notgenetically related.

18. The vertebrate, cell or method of preceding paragraph, wherein atleast one of the different segments is a synthetic mutant of a humangermline gene segment.

19. The vertebrate, cell or method of any preceding paragraph, whereineach of said gene segments occurs in 10 or more different humanpopulations.

20. The vertebrate, cell or method of preceding paragraph, wherein eachof said gene segments has a human frequency of 5% or greater (eg, 10,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% orgreater).

In this respect, the skilled person can be guided by the informationprovided in Table 14. Frequency can, for example, be cumulativefrequency in the 1000 Genomes database.

21. The vertebrate, cell or method of paragraph 20, wherein each of saidgene segments occurs in 10 or more different human populations.

22. The vertebrate, cell or method of any preceding paragraph, whereineach of said gene segments occurs in the 1000 Genomes database in morethan 50 individuals.

23. The vertebrate, cell or method of preceding paragraph, wherein eachof said gene segments (i) has a human frequency of 5% or greater (eg,10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%or greater); and (ii) occurs in 10 or more different human populations.

In this respect, the skilled person can be guided by the informationprovided in Table 14.

Frequency can, for example, be cumulative frequency in the 1000 Genomesdatabase.

24. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingfirst and second human Ig locus gene segments of the same type (eg,first and second human JH6 gene segments; or first and second IgG2 genesegments; or first and second human Jλ7 gene segments), wherein thefirst gene segment is a gene segment selected from Table 14 (eg,IGHJ6-a) and the second gene segment is the corresponding referencesequence (eg, IGHJ6 ref; SEQ ID NO: 244).

Table 14 lists commonly-occurring natural human variants. It can be seenthat these occur across many human populations and thus usefully havewide applicability for human antibody-based drugs.

For example, the gene segments are provided as targeted insertions intoan endogenous non-human vertebrate Ig locus. Alternatively, randomintegration (eg, using YACs) as is know in the art can be performed.

For example, the genome comprises a variable region that comprises V, Dand J gene segments (for the variable region of a heavy chain locus) orV and J gene segments (for the variable region of a light chain locus)upstream of a constant region for expression of heavy or light chainsrespectively.

In another embodiment, the invention enables the skilled person toselect two or more different naturally-occurring human gene segmentvariants for combination into the genome of a non-human vertebrate orcell. A reference sequence need not be included. It may be desirable touse one or more rare gene segments to increase diversity of therepertoire. Additionally or alternatively, it may be desirable toinclude a mixture of frequent and rare variants of the same type toprovide repertoire diversity. The variants may be chosen additionally oralternatively to tailor the gene segment inclusion to one or morespecific human populations as indicated by the information provided inTable 13 or Table 14.

Thus, the invention provides

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising first andsecond human Ig locus gene segments of the same type (eg, first andsecond human JH6 gene segments; or first and second IgG2 gene segments;or first and second human Jλ7 gene segments), wherein the gene segmentsare gene segments selected from Table 13 or Table 14; and optionallywherein one or more of the gene segments appears in Table 14 (eg,IGHJ6-a) or is a reference sequence (eg, IGHJ6 ref; SEQ ID NO: 244).

25. The vertebrate or cell of paragraph 24, wherein the genome comprisesa third human gene segment of said type, the third gene segment beingdifferent from the first and second gene segments.

26. The vertebrate or cell of paragraph 24 or 25, wherein the first andsecond gene segments are cis on the same chromosome; and optionally thethird gene segment is also cis on said chromosome.

27. The vertebrate or cell of paragraph 26, wherein the gene segmentsare targeted insertions into an endogenous non-human Ig locus.

For example, the gene segments are heavy chain gene segments and thenon-human locus is an IgH locus. For example, the gene segments arelight chain (kappa or lambda) gene segments and the non-human locus isan IgL locus.

28. The vertebrate or cell of paragraph 24 or 25, wherein the first andsecond gene segments are trans on different chromosomes.

Thus, the chromosomes are the same type (eg, both mouse chromosome 6 orrat chromosome 4).

29. The vertebrate or cell of any one of paragraphs 24 to 28, whereinthe first gene segment is a gene segment selected from any one of Tables1 to 7 and 9 to 14 (eg, selected from Table 13 or 14) and the secondgene segment is the corresponding reference sequence.

30. A population of non-human vertebrates (eg, mice or rats) comprisingfirst and second human Ig locus gene segments of the same type (eg,first and second human JH6 gene segments; or first and second IgG2 genesegments; or first and second human Jλ7 gene segments), wherein thefirst gene segment is a gene segment selected from any one of Tables 1to 7 and 9 to 14 (eg, Table 13 or 14) (eg, IGHJ6-a) and the second genesegment is the corresponding reference sequence (eg, SEQ ID NO: 7),wherein the first gene segment is provided in the genome of a firstvertebrate of the population, and the second gene segment is provided inthe genome of a second vertebrate of the population.

31. The population of paragraph 30, wherein the genome of the firstvertebrate does not comprise the second gene segment.

32. The population of paragraph 30 or 31, wherein the populationcomprises a third human gene segment of said type, the third genesegment being different from the first and second gene segments andoptionally wherein the first and third gene segments are present in thegenome of the first vertebrate.

33. The population of paragraph 30, 31 or 32, wherein the gene segmentsare targeted insertions into an endogenous non-human Ig locus in therespective genome.

For example, the gene segments are heavy chain gene segments and thenon-human locus is an IgH locus. For example, the gene segments arelight chain (kappa or lambda) gene segments and the non-human locus isan IgL locus.

34. The population of any one of paragraphs 30 to 33, wherein the firstgene segment is a gene segment selected from any one of Tables 1 to 7and 9 to 14 (eg, Table 13 or 14) and the second gene segment is thecorresponding reference sequence.

35. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising first and second humanIg locus gene segments of the same type (eg, first and second human JH6gene segments; or first and second IgG2 gene segments; or first andsecond human Jλ7 gene segments), wherein the first gene segment is agene segment selected from any one of Tables 1 to 7 and 9 to 14 (eg,Table 13 or 14) (eg, IGHJ6-a) and the second gene segment is thecorresponding reference sequence (eg, SEQ ID NO: 7).

36. A method of providing an enhanced human immunogolobulin gene segmentrepertoire, the method comprising providing a population according toany one of paragraphs 30 to 33.

Variants Prevalent in Few Populations

In another aspect, it is of note that certain human gene segmentvariants may appear relatively frequently in one or a small number ofpopulations, but is not found prevalently across many different humanpopulations. There is thinking that specific germline gene segmentrepertoires have evolved in individual human ethnic populations due toiterative exposure to antigens (eg, disease pathogen antigens) to whichthe population is often exposed. Repeated exposure and mutation may havelead to the evolution of gene segment variants that can provide aneffective response to the antigen (pathogen) in the population, and thismay explain the conservation of the gene segments in those populations(as opposed to other human ethnic populations that may not havefrequently encountered the antigen). With this in mind, the inventorsidentified gene segment variants from their analysis that are relativelyprevalent in a small number of human populations, and not across manypopulations. The inventors realized that inclusion of one or more ofsuch gene segments in the configurations of the invention (eg, intransgenic Ig loci, vertebrates and cells) would be useful for producingantibodies, Ig chains and variable domains that can address antigens(eg, disease-causing antigens or pathogens) to which the small number ofhuman populations may become exposed. Such products would be useful fortreating and/or preventing disease or medical conditions in members ofsuch a population. This aspect could also be useful for addressinginfectious disease pathogens that may have been common in the smallnumber of populations, but which in the future or relatively recently inevolution has become a more prevalent disease-causing pathogen in otherhuman populations (ie, those not listed in Table 13 against the genesegment variant(s) in question). To this end, from the 1000 Genomesdatabase the inventors have identified the gene segment variants listedin Table 20.

Thus, according to any configuration or aspect described herein, one,more or all of the gene segments used in the present invention can be agene segment listed in Table 20A, 20B, 20C or 20D.

Multiple JH Gene Segment Variants

A specific application of this configuration is the provision ofmultiple human JH gene segments as follows.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 3human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 orJH6), wherein at least two of the human JH gene segments are variantsthat are not identical to each other.

In an example, any cell of the invention is an isolated cell. An“isolated” cell is one that has been identified, separated and/orrecovered from a component of its production environment (eg, naturallyor recombinantly). Preferably, the isolated cell is free of associationwith all other components from its production environment, eg, so thatthe cell can produce an antibody to an FDA-approvable or approvedstandard. Contaminant components of its production environment, such asthat resulting from recombinant transfected cells, are materials thatwould typically interfere with research, diagnostic or therapeutic usesfor the resultant antibody, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In preferred embodiments,the polypeptide will be purified: (1) to greater than 95% by weight ofantibody as determined by, for example, the Lowry method, and in someembodiments, to greater than 99% by weight; (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under non-reducing or reducing conditions using Coomassie blueor, preferably, silver stain. Ordinarily, however, an isolated cell willbe prepared by at least one purification step.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different non-endogenous JH gene segments (eg, human gene segments) ofthe same type (JH1, JH2, JH3, JH4, JH5 or JH6) cis at the same Ig (eg,IgH, eg, endogenous IgH, eg, mouse or rat IgH) locus. In an example, thegenome comprises a human VH, D and JH repertoire comprising saiddifferent JH gene segments. Optionally the non-endogenous JH genesegments are non-mouse or non-rat, eg, human JH gene segments. In anexample one or more or all of the non-endogenous gene segments aresynthetic.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different human JH gene segments of the same type (JH1, JH2, JH3, JH4,JH5 or JH6) trans at the same Ig (eg, IgH, eg, endogenous IgH, eg, mouseor rat IgH) locus; and optionally a third human JH gene segments of thesame type, wherein the third JH is cis with one of said 2 different JHgene segments.

A population of non-human vertebrates (eg, mice or rats) comprising arepertoire of human JH gene segments, wherein the plurality comprises atleast 2 different human JH gene segments of the same type (JH1, JH2,JH3, JH4, JH5 or JH6), a first of said different JH gene segments isprovided in the genome of a first vertebrate of the population, and asecond of said different JH gene segments being provided in the genomeof a second vertebrate of the population, wherein the genome of thefirst vertebrate does not comprise the second JH gene segment.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebratecell (eg, an ES cell or a B-cell) having a genome comprising at least 2different non-endogenous (eg, human) JH gene segments of the same type(JH1, JH2, JH3, JH4, JH5 or JH6), wherein the JH gene segments arederived from the genome sequence of different human individuals that arenot genetically related over at least 3 generations (eg, 3, 4, 5 or 6generations). Optionally the non-endogenous JH gene segments are humanJH gene segments. In an example one or more or all of the non-endogenousgene segments are synthetic.

A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 3 human JHgene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), whereinat least two of the human JH gene segments are variants that are notidentical to each other.

A method of enhancing the immunoglobulin gene diversity of a non-humanvertebrate (eg, a mouse or rat), the method comprising providing thevertebrate with a genome comprising at least 2 different non-endogenous(eg, human) JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5or JH6) cis at the same Ig (eg, IgH, eg, endogenous IgH, eg, mouse orrat IgH) locus). Optionally the non-endogenous JH gene segments arenon-mouse or non-rat, eg, human JH gene segments. In an example one ormore or all of the non-endogenous gene segments are synthetic.

A method of enhancing the immunoglobulin gene diversity of a non-humanvertebrate (eg, a mouse or rat), the method comprising providing thevertebrate with a genome comprising at least 2 different human JH genesegments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6) trans at thesame Ig (eg, IgH, eg, endogenous IgH, eg, mouse or rat IgH) locus; andoptionally a third human JH gene segments of the same type, wherein thethird JH is cis with one of said 2 different JH gene segments.

A method of providing an enhanced human immunoglobulin JH gene segmentrepertoire, the method comprising providing a population of non-humanvertebrates (eg, a mouse or rat) comprising a repertoire of human JHgene segments, wherein the method comprises providing at least 2different human JH gene segments of the same type (JH1, JH2, JH3, JH4,JH5 or JH6), wherein a first of said different JH gene segments isprovided in the genome of a first vertebrate of the population, and asecond of said different JH gene segments is provided in the genome of asecond vertebrate of the population, wherein the genome of the firstvertebrate does not comprise the second JH gene segment.

A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous (eg, human) JH gene segments of the same type (JH1, JH2,JH3, JH4, JH5 or JH6), wherein the JH gene segments are derived from thegenome sequence of different human individuals that are not geneticallyrelated over at least 3 generations (eg, 3, 4, 5, or 6 generations).Optionally the non-endogenous JH gene segments are human JH genesegments. In an example one or more or all of the non-endogenous genesegments are synthetic.

In an example of the vertebrate or cell or the method of the inventionat least 2 or 3 of said different gene segments are provided cis at thesame Ig locus in said genome.

In an example of the vertebrate or cell or the method of the inventionthe JH gene segments are derived from the genome sequence of differenthuman individuals that are not genetically related over at least 3generations (eg, 3, 4, 5, or 6 generations).

In an example of the vertebrate or cell or the method of the inventionthe JH gene segments are derived from the genome sequence of two or moredifferent human individuals; optionally wherein the different humanindividuals are from different human populations.

In an example of the vertebrate or cell or the method of the inventionthe individuals are not genetically related (eg, going back 3, 4, 5, or6 generations).

In an example of the vertebrate or cell or the method of the inventionat least one of the different JH segments is a synthetic mutant of ahuman germline JH gene segment.

The invention also provides a method of enhancing the humanimmunoglobulin gene diversity of a non-human vertebrate (eg, a mouse orrat), the method comprising providing the vertebrate with a genomecomprising at least 2 human JH gene segments of the same type (JH1, JH2,JH3, JH4, JH5 or JH6), wherein the JH gene segments are derived from thegenome sequence of different human individuals that are not geneticallyrelated over at least 3 generations (eg, 3, 4, 5, or 6 generations);optionally wherein at least 2 or 3 of said different gene segments areprovided at the same IgH locus in said genome.

In an example of the vertebrate or cell or the method of this embodimentof the invention the genome comprises a substantially completefunctional repertoire of human JH gene segment types supplemented withone, two or more human JH gene segments, wherein said substantiallycomplete functional repertoire and the supplementary JH gene segmentsare not found together in the germline genome of a human individual.

In an example of the population of the invention, the populationcomprises a substantially complete functional repertoire of human JHgene segment types supplemented with one, two or more human JH genesegments, wherein said substantially complete functional repertoire andthe supplementary JH gene segments are not found together in thegermline genome of a human individual.

A non-human vertebrate (eg, a mouse or rat) or a non-human cell (eg, anES cell or a B-cell) having a genome comprising a substantially completefunctional repertoire of human JH gene segment types supplemented withone, two or more human JH gene segments, wherein said substantiallycomplete functional repertoire and the supplementary JH gene segmentsare not found together in the germline genome of a human individual.

A population of non-human vertebrates (eg, mice or rats) comprising asubstantially complete functional repertoire of human JH gene segmenttypes supplemented with one, two or more human JH gene segments, whereinsaid substantially complete functional repertoire and the supplementaryJH gene segments are not found together in the germline genome of ahuman individual.

In an example of the vertebrate or the population, at least one of saidJH gene segments is SEQ ID NO: 1, 2, 3 or 4. For example, at least oneof said JH gene segments is SEQ ID NO: 1 and at least one, two or moreof said supplementary JH gene segments is a variant according to anyexample above. For example, at least one of said JH gene segments is SEQID NO: 2 and at least one, two or more of said supplementary JH genesegments is a variant according to any one of the examples above. Forexample, at least one of said JH gene segments is SEQ ID NO: 2 and atleast one, two or more of said supplementary JH gene segments is avariant according to any one of the examples above.

In an embodiment, the non-human vertebrate or vertebrate cell of theinvention comprises a genome that comprises VH, D and JH generepertoires comprising human gene segments, the JH gene repertoire (eg,a human JH gene segment repertoire) comprising a plurality of JH1 genesegments provided by at least 2 different JH1 gene segments in cis atthe same Ig locus in said genome;

a plurality of JH2 gene segments provided by at least 2 different JH2gene segments in cis at the same Ig locus in said genome;

a plurality of JH3 gene segments provided by at least 2 different JH3gene segments in cis at the same Ig locus in said genome;

a plurality of JH4 gene segments provided by at least 2 different JH4gene segments in cis at the same Ig locus in said genome;

a plurality of JH5 gene segments provided by at least 2 different JH5gene segments in cis at the same Ig locus in said genome; and/or

a plurality of JH6 gene segments provided by at least 2 different JH6gene segments in cis at the same Ig locus in said genome;

optionally wherein the JH gene segments are derived from the genomesequence of two or more different human individuals.

Optionally said at least 2 different JH gene segments are human genesegments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a IgH locus, eg, an endogenous locus, eg, amouse or rat IgH locus.

In an embodiment, the non-human vertebrate or vertebrate cell of theinvention comprises a genome that comprises VH, D and JH generepertoires comprising human gene segments, the JH gene repertoire (eg,a human JH gene segment repertoire) comprising a plurality of JH1 genesegments provided by at least 3 different JH1 gene segments; a pluralityof JH2 gene segments provided by at least 3 different JH2 gene segments;a plurality of JH3 gene segments provided by at least 3 different JH3gene segments; a plurality of JH4 gene segments provided by at least 3different JH4 gene segments; a plurality of JH5 gene segments providedby at least 3 different JH5 gene segments; and/or a plurality of JH6gene segments provided by at least 3 different JH6 gene segments;optionally wherein the JH gene segments are derived from the genomesequence of two or three different human individuals;

optionally wherein at least 2 or 3 of said different gene segments areprovided in cis at the same Ig locus in said genome.

Optionally said at least 3 different JH gene segments are human genesegments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a IgH locus, eg, an endogenous locus, eg, amouse or rat IgH locus.

Optionally in the vertebrate or cell the different human individuals arefrom different human populations.

Optionally in the vertebrate or cell the individuals are not geneticallyrelated (eg, Going back 3, 4, 5 or 6 generations).

Optionally in the vertebrate or cell at least one of the different JHsegments is a synthetic mutant of a human germline JH gene segment.

In an embodiment of a non-human vertebrate or vertebrate cell(optionally an ES cell or B-cell) according to the invention, thevertebrate or cell genome comprises human VH, D and JH gene repertoires,the JH gene repertoire (eg, a human JH gene repertoire) comprising aplurality of JH1 gene segments provided by at least 2 different humanJH1 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of JH2 gene segments provided by at least 2 different humanJH2 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of JH3 gene segments provided by at least 2 different humanJH3 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of JH4 gene segments provided by at least 2 different humanJH4 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of JH5 gene segments provided by at least 2 different humanJH5 gene segments, optionally in cis at the same Ig locus in saidgenome; and/or

a plurality of JH6 gene segments provided by at least 2 different humanJH6 gene segments, optionally in cis at the same Ig locus in saidgenome;

wherein the JH gene segments are derived from the genome sequence ofdifferent human individuals that are not genetically related over atleast 3 generations (eg, 3, 4, 5 or 6 generations).

Optionally said at least 2 different JH gene segments are human genesegments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a IgH locus, eg, an endogenous locus, eg, amouse or rat IgH locus. Optionally in the vertebrate or cell the humanindividuals are from different human populations.

JH5

An embodiment provides a vertebrate, cell or population of the inventionwhose genome comprises a plurality of JH5 gene segments, wherein theplurality comprises a human JH5 gene variant of SEQ ID NO: 1, whereinthe variant comprises a nucleotide mutation at one or more positionscorresponding to positions

106,330,024

106,330,027

106,330,032

106,330,041

106.330.44

106.330.45

106.330.62

106.330.63

106.330.65

106.330.66

106.330.67

106.330.68 and

106,330,071

on human chromosome 14.

In the vertebrate, cell or population optionally the plurality comprisesa human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprisesa guanine at a position corresponding to position 106,330,067 on humanchromosome 14; and optionally no further mutation from the sequence ofSEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106,330,071 on human chromosome 14(optionally the additional mutation being a guanine); (ii) position106,330,066 on human chromosome 14 (optionally the additional mutationbeing a guanine); and/or (iii) position 106,330,068 on human chromosome14 (optionally the additional mutation being a thymine).

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a guanine at a positioncorresponding to position 106,330,071 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106,330,063 on human chromosome 14(optionally the additional mutation being an adenine); and/or (ii)position 106,330,067 on human chromosome 14 (optionally the additionalmutation being a guanine).

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a cytosine at a positioncorresponding to position 106,330,045 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises an adenine at a positioncorresponding to position 106,330,044 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106.330.66 on human chromosome 14(optionally the additional mutation being a guanine); and/or (ii)position 106,330,068 on human chromosome 14 (optionally the additionalmutation being a thymine).

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a guanine at a positioncorresponding to position 106,330,066 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106.330.67 on human chromosome 14(optionally the additional mutation being a guanine);

and/or (ii) position 106,330,068 on human chromosome 14 (optionally theadditional mutation being a thymine).

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a thymine at a positioncorresponding to position 106,330,068 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106,330,067 on human chromosome 14(optionally the additional mutation being a guanine); and/or (ii)position 106,330,066 on human chromosome 14 (optionally the additionalmutation being a guanine).

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a cytosine at a positioncorresponding to position 106,330,027 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises an adenine at a positioncorresponding to position 106,330,024 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a thymine at a positioncorresponding to position 106,330,032 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a thymine at a positioncorresponding to position 106,330,041 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises an adenine or thymine at a positioncorresponding to position 106,330,063 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a positioncorresponding to position 106,330,071 on human chromosome 14 (optionallythe additional mutation being a guanine).

Optionally the plurality comprises a human JH5 gene variant of SEQ IDNO: 1, wherein the variant comprises a cytosine at a positioncorresponding to position 106,330,062 on human chromosome 14; andoptionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the genome comprises SEQ ID NO:1; optionally in cis at thesame Ig locus as one, two or more of the variants.

JH6

An embodiment provides a vertebrate, cell or population of the inventionwhose genome comprises a plurality of JH6 gene segments, wherein theplurality comprises a human JH6 gene variant of SEQ ID NO: 2, whereinthe variant comprises a nucleotide mutation at one or more positionscorresponding to positions

106,329,411

106.329.413

106.329.414

106,329,417

106,329,419

106,329,426

106,329,434

106,329,435, and

106,329,468

on human chromosome 14.

Optionally the genome of the vertebrate, cell or population comprises aplurality of JH6 gene segments, wherein the plurality comprises a humanJH6 gene variant of SEQ ID NO: 2, wherein the variant comprises aguanine at a position corresponding to position 106,329,435 on humanchromosome 14; and optionally no further mutation from the sequence ofSEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106,329,468 on human chromosome 14(optionally the additional mutation being a guanine); (ii) position106,329,419 on human chromosome 14 (optionally the additional mutationbeing an adenine); (iii) position 106,329,434 on human chromosome 14(optionally the additional mutation being a cytosine) and/or position106,329,414 on human chromosome 14 (optionally the additional mutationbeing a guanine); (iv) position 106,329,426 on human chromosome 14(optionally the additional mutation being an adenine); (v) position106,329,413 on human chromosome 14 (optionally the additional mutationbeing an adenine); (vi) position 106,329,417 on human chromosome 14(optionally the additional mutation being a thymine); (vii) position106,329,411 on human chromosome 14 (optionally the additional mutationbeing a thymine); (viii) position 106,329,451 on human chromosome 14(optionally the additional mutation being an adenine); (ix) position106,329,452 on human chromosome 14 (optionally the additional mutationbeing a cytosine); and/or (x) position 106,329,453 on human chromosome14 (optionally the additional mutation being a cytosine).

Optionally the variant comprises additionally mutations at positionscorresponding to position 106.329.451 on human chromosome 14, theadditional mutation being an adenine; position 106.329.452 on humanchromosome 14, the additional mutation being a cytosine; and position106.329.453 on human chromosome 14, the additional mutation being acytosine.

The vertebrate, cell or population optionally comprises a plurality ofJH6 gene segments, wherein the plurality comprises a human JH6 genevariant of SEQ ID NO: 2, wherein the variant comprises a guanine at aposition corresponding to position 106,329,468 on human chromosome 14;and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a positioncorresponding to position 106,329,435 on human chromosome 14 (optionallythe additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality ofJH6 gene segments, wherein the plurality comprises a human JH6 genevariant of SEQ ID NO: 2, wherein the variant comprises a thymine at aposition corresponding to position 106,329,417 on human chromosome 14;and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a positioncorresponding to position 106,329,435 on human chromosome 14 (optionallythe additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality ofJH6 gene segments, wherein the plurality comprises a human JH6 genevariant of SEQ ID NO: 2, wherein the variant comprises a cytosine at aposition corresponding to position 106,329,434 on human chromosome 14;and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106,329,414 on human chromosome 14(optionally the additional mutation being a guanine); and/or (ii)position 106,329,435 on human chromosome 14 (optionally the additionalmutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality ofJH6 gene segments, wherein the plurality comprises a human JH6 genevariant of SEQ ID NO: 2, wherein the variant comprises a thymine at aposition corresponding to position 106,329,411 on human chromosome 14;and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a positioncorresponding to position 106,329,435 on human chromosome 14 (optionallythe additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality ofJH6 gene segments, wherein the plurality comprises a human JH6 genevariant that is an antisense sequence of a variant described above.

Optionally the genome comprises SEQ ID NO:2; optionally cis at the sameIg locus as one, two or more of the JH6 variants.

JH2

An embodiment provides a vertebrate, cell or population of the inventionwhose genome comprises a plurality of JH2 gene segments, wherein theplurality comprises a human JH2 gene variant of SEQ ID NO: 3, whereinthe variant comprises a nucleotide mutation at one or more positionscorresponding to positions

106,331,455

106,331,453, and

106,331,409

on human chromosome 14.

Optionally the vertebrate, cell or population comprises said pluralityof JH2 gene segments, wherein the plurality comprises a human JH2 genevariant of SEQ ID NO: 3, wherein the variant comprises a guanine at aposition corresponding to position 106,331,455 on human chromosome 14;and optionally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the variant comprises additionally a mutation at a positioncorresponding to (i) position 106,331,453 on human chromosome 14(optionally the additional mutation being an adenine); and/or (ii)position 106,331,409 on human chromosome 14 (optionally the additionalmutation being an adenine); (iii) position 106,329,434 on humanchromosome 14 (optionally the additional mutation being an adenine).

Optionally the vertebrate, cell or population comprises a plurality ofJH2 gene segments, wherein the plurality comprises a human JH2 genevariant of SEQ ID NO: 3, wherein the variant comprises an adenine at aposition corresponding to position 106,331,453 on human chromosome 14;and optionally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the variant comprises additionally a mutation at a positioncorresponding to position 106,331,409 on human chromosome 14 (optionallythe additional mutation being an adenine).

Optionally the vertebrate, cell or population comprises a plurality ofJH2 gene segments, wherein the plurality comprises a human JH2 genevariant of SEQ ID NO: 3, wherein the variant comprises an adenine at aposition corresponding to position 106,331,409 on human chromosome 14;and optionally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the vertebrate, cell or population comprises a plurality ofJH2 gene segments, wherein the plurality comprises a human JH2 genevariant that is an antisense sequence of a variant described above.

Optionally the genome comprises SEQ ID NO:3; optionally cis at the sameIg locus as one, two or more of the JH2 variants.

Optionally the vertebrate, cell or population genome comprises two ormore different JH gene segments selected from SEQ ID NOs: 1 to 3 andvariants described above; optionally wherein said JH gene segments arecis at the same immunoglobulin Ig locus.

Multiple Human D Gene Segment Variants

A specific application of this configuration is the provision ofmultiple human D gene segments as follows (as set out in numberedclauses, starting at clause number 154).

154. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 3 human D gene segments of the same type (eg, D2-2 genesegments), wherein at least two of the human D gene segments arevariants that are not identical to each other (eg, D2-2ref and D2-2a).

In an example of any aspect of the sixth configuration of the invention(V, D, J or C), one or more or all of the variants arenaturally-occurring human gene segments.

In an example of any aspect of the sixth configuration of the invention(V, D, J or C), one or more of the variants may be a synthetic variantof a human gene segment.

155. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 2 different non-endogenous D gene segments of the same type(eg, D2-2ref and D2-2a) cis at the same Ig locus.

156. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 2 different human D gene segments of the same type (eg, D2-2refand D2-2a) trans at the same Ig locus; and optionally a third human Dgene segment (eg, (eg, D2-2ref, D2-2a or D2-2b) of the same type,wherein the third D is cis with one of said 2 different D gene segments.

157. A population of non-human vertebrates (eg, mice or rats) comprisinga repertoire of human D gene segments, wherein the plurality comprisesat least 2 different human D gene segments of the same type (eg, D2-2gene segments), a first of said different D gene segments (eg, D2-2ref)is provided in the genome of a first vertebrate of the population, and asecond of said different D gene segment (eg, D2-2a) being provided inthe genome of a second vertebrate of the population, wherein the genomeof the first vertebrate does not comprise the second D gene segment.

158. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 2 different non-endogenous D gene segments of the same type(eg, human D2-2 gene segments), wherein the D gene segments are derivedfrom the genome sequence of different human individuals that are notgenetically related over at least 3 generations.

159. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 3 human Dgene segments of the same type (eg, D2-2 gene segments), wherein atleast two of the human D gene segments are variants that are notidentical to each other (eg, D2-2ref and D2-2a).

160. A method of enhancing the immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous D gene segments of the same type (eg, human D2-2 genesegments) cis at the same Ig locus.

161. A method of enhancing the immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differenthuman D gene segments of the same type (eg, D2-2ref and D2-2a) trans atthe same Ig locus; and optionally a third human D gene segment (eg,D2-2ref, D2-2a or D2-2b) of the same type, wherein the third D is ciswith one of said 2 different D gene segments.

162. A method of providing an enhanced human immunoglobulin D genesegment repertoire, the method comprising providing a population ofnon-human vertebrates (eg, a mouse or rat) comprising a repertoire ofhuman D gene segments, wherein the method comprises providing at least 2different human D gene segments of the same type (eg, D2-2ref andD2-2a), wherein a first of said different D gene segments is provided inthe genome of a first vertebrate of the population, and a second of saiddifferent D gene segments is provided in the genome of a secondvertebrate of the population, wherein the genome of the first vertebratedoes not comprise the second D gene segment.

163. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous D gene segments of the same type (eg, D2-2ref and D2-2a),wherein the D gene segments are derived from the genome sequence ofdifferent human individuals that are not genetically related over atleast 3 generations.

164. The vertebrate or cell of clause 154, 156 or 158, or the method ofclause 159, 161 or 163, wherein at least 2 or 3 of said different genesegments are provided cis at the same Ig locus in said genome.

165. The vertebrate or cell of clause 154, 155 or 156, or the method ofany one of clauses 159 to 162 and 164, wherein the D gene segments arederived from the genome sequence of different human individuals that arenot genetically related over at least 3 generations.

166. The vertebrate or cell of any one of clauses 154 to 157, or themethod of any one of clauses 159 to 162 and 165, wherein the D genesegments are derived from the genome sequence of two or more differenthuman individuals; optionally wherein the different human individualsare from different human populations.

167. The vertebrate, cell or method of clause 166, wherein theindividuals are not genetically related.

168. The vertebrate, cell or method of any one of clauses 154 to 167,wherein at least one of the different D segments is a synthetic mutantof a human germline D gene segment.

169. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 human Dgene segments of the same type (eg, D2-2ref and D2-2a), wherein the Dgene segments are derived from the genome sequence of different humanindividuals that are not genetically related over at least 3generations; optionally wherein at least 2 or 3 of said different genesegments are provided at the same IgH locus in said genome.

170. The vertebrate or cell of any one of clauses 154 to 158 and 164 to168, wherein the genome comprises a substantially complete functionalrepertoire of human D gene segment types supplemented with one, two ormore variant human D gene segments, wherein said substantially completefunctional repertoire and the supplementary D gene segments are notfound together in the germline genome of a human individual.

171. The population of clause 157, wherein the population comprises asubstantially complete functional repertoire of human D gene segmenttypes supplemented with one, two or more variant human D gene segments,wherein said substantially complete functional repertoire and thesupplementary D gene segments are not found together in the germlinegenome of a human individual.

172. A non-human vertebrate (eg, a mouse or rat) or a non-human cell(eg, an ES cell or a B-cell) having a genome comprising a substantiallycomplete functional repertoire of human D gene segment typessupplemented with one, two or more variant human D gene segments,wherein said substantially complete functional repertoire and thesupplementary D gene segments are not found together in the germlinegenome of a human individual.

173. A population of non-human vertebrates (eg, mice or rats) comprisinga substantially complete functional repertoire of human JH gene segmenttypes supplemented with one, two or more variant human D gene segments,wherein said substantially complete functional repertoire and thesupplementary D gene segments are not found together in the germlinegenome of a human individual.

174. The vertebrate or cell of clause 172 or the population of clause173, comprising first and second D gene segments selected from D2-2refand D2-2a; or D2-21 ref and D2-21a; or D3-10ref and D3-10a; or D3-16refand D3-16a; or D2-8ref and D2-8a; or D3-3ref and D3-3a; or D4-23ref andD4-23a; or D6-13ref and D6-13a; or D3-9ref and D3-9a; or D4-4ref andD4-4a; or D7-27ref and D7-27a;

Optionally wherein the first and/or second D gene segment is present intwo or more copies.

For example, there are provided two or three copies of the first genesegment, optionally with one, two or three copies of the second genesegment. Copies can be arranged in cis or trans.

175. The vertebrate, cell or population of clause 174, comprising humangene segments D2-2ref and D2-2a; and D3-3ref and D3-3a; and optionallyalso D2-15.

In an example, the vertebrate, cell or population comprises one or moreD segments selected from human D3-3, D2-15, D3-9; D4-17; D3-10; D2-2;D5-24; D6-19; D3-22; D6-13; D5-12; D1-26; D1-20; D5-18; D3-16; D2-21;D1-14; D7-27; D1-1; D6-25; D2-14 and D4-23 (eg, selected from D3-9*01;D4-17*01; D3-10*01; D2-2*02; D5-24*01; D6-19*01; D3-22*01; D6-13*01;D5-12*01; D1-26*01; D1-20*01; D5-18*01; D3-16*02; D2-21*02; D1-14*01;D7-27*02; D1-1*01; D6-25*01; D2-15*01; and D4-23*01), together with thereference sequence(s) of said selected segment(s). These were found invariable domains having a HCDR3 length of at least 20 amino acids (seeexamples herein).

176. A non-human vertebrate or vertebrate cell according to clause 155,comprising a genome that comprises VH, D and JH gene repertoirescomprising human gene segments, the D gene repertoire comprising one ormore of

a plurality of D2-2 gene segments provided by at least 2 different D2-2gene segments in cis at the same Ig locus in said genome;

a plurality of D2-21 gene segments provided by at least 2 differentD2-21 gene segments in cis at the same Ig locus in said genome;

a plurality of D3-10 gene segments provided by at least 2 differentD3-10 gene segments in cis at the same Ig locus in said genome;

a plurality of D3-16 gene segments provided by at least 2 differentD3-16 gene segments in cis at the same Ig locus in said genome;

a plurality of D2-8 gene segments provided by at least 2 different D2-8gene segments in cis at the same Ig locus in said genome;

a plurality of D3-3 gene segments provided by at least 2 different D3-3gene segments in cis at the same Ig locus in said genome;

a plurality of D4-23 gene segments provided by at least 2 differentD4-23 gene segments in cis at the same Ig locus in said genome;

a plurality of D6-13 gene segments provided by at least 2 differentD6-13 gene segments in cis at the same Ig locus in said genome;

a plurality of D3-9 gene segments provided by at least 2 different D3-9gene segments in cis at the same Ig locus in said genome;

a plurality of D4-4 gene segments provided by at least 2 different D4-4gene segments in cis at the same Ig locus in said genome; and

a plurality of D7-27 gene segments provided by at least 2 differentD7-27 gene segments in cis at the same Ig locus in said genome;

optionally wherein the D gene segments are derived from the genomesequence of two or more different human individuals.

177. A non-human vertebrate or vertebrate cell according to clause 155,comprising a genome that comprises VH, D and JH gene repertoirescomprising human gene segments, the D gene repertoire comprising one ormore of

a plurality of D2-2 gene segments provided by at least 2 different D2-2gene segments in trans in said genome;

a plurality of D2-21 gene segments provided by at least 2 differentD2-21 gene segments in trans in said genome;

a plurality of D3-10 gene segments provided by at least 2 differentD3-10 gene segments in trans in said genome;

a plurality of D3-16 gene segments provided by at least 2 differentD3-16 gene segments in trans in said genome;

a plurality of D2-8 gene segments provided by at least 2 different D2-8gene segments in trans in said genome;

a plurality of D3-3 gene segments provided by at least 2 different D3-3gene segments in trans in said genome;

a plurality of D4-23 gene segments provided by at least 2 differentD4-23 gene segments in trans in said genome;

a plurality of D6-13 gene segments provided by at least 2 differentD6-13 gene segments in trans in said genome;

a plurality of D3-9 gene segments provided by at least 2 different D3-9gene segments in trans in said genome;

a plurality of D4-4 gene segments provided by at least 2 different D4-4gene segments in trans in said genome; and

a plurality of D7-27 gene segments provided by at least 2 differentD7-27 gene segments in trans in said genome;

optionally wherein the D gene segments are derived from the genomesequence of two or more different human individuals.

178. A non-human vertebrate or vertebrate cell (optionally an ES cell orB-cell), according to clause 154, comprising a genome that comprises VH,D and JH gene repertoires comprising human gene segments, the D generepertoire comprising one or more of a plurality of D2-2 gene segmentsprovided by at least 3 different D2-2 gene segments; a plurality ofD2-21 gene segments provided by at least 3 different D2-21 genesegments; a plurality of D3-10 gene segments provided by at least 3different D3-10 gene segments; a plurality of D3-16 gene segmentsprovided by at least 3 different D3-16 gene segments; a plurality ofD2-8 gene segments provided by at least 3 different D2-8 gene segments;a plurality of D3-3 gene segments provided by at least 3 different D3-3gene segments; a plurality of D4-23 gene segments provided by at least 3different D4-23 gene segments; a plurality of D6-13 gene segmentsprovided by at least 3 different D6-13 gene segments; a plurality ofD3-9 gene segments provided by at least 3 different D3-9 gene segments;a plurality of D4-4 gene segments provided by at least 3 different D4-4gene segments; and a plurality of D7-27 gene segments provided by atleast 3 different D7-27 gene segments;

optionally wherein the D gene segments are derived from the genomesequence of two or three different human individuals;

optionally wherein at least 2 or 3 of said different gene segments areprovided in cis at the same Ig locus in said genome.

179. The vertebrate or cell of clause 176, 177 or 178, wherein thedifferent human individuals are from different human populations.

180. The vertebrate or cell of any one of clauses 176 to 179, whereinthe individuals are not genetically related.

181. The vertebrate or cell of any one of clauses 176 to 180, wherein atleast one of the different D segments is a synthetic mutant of a humangermline D gene segment.

182. A non-human vertebrate or vertebrate cell (optionally an ES cell orB-cell) according to clause 158, comprising a genome comprising humanVH, D and JH gene repertoires, the D gene repertoire comprising of oneor more of a plurality of D2-2 gene segments provided by at least 2different D2-2 gene; optionally in cis in said genome;

a plurality of D2-21 gene segments provided by at least 2 differentD2-21 gene; optionally in cis in said genome;

a plurality of D3-10 gene segments provided by at least 2 differentD3-10 gene; optionally in cis in said genome;

a plurality of D3-16 gene segments provided by at least 2 differentD3-16 gene; optionally in cis in said genome;

a plurality of D2-8 gene segments provided by at least 2 different D2-8gene; optionally in cis in said genome;

a plurality of D3-3 gene segments provided by at least 2 different D3-3gene; optionally in cis in said genome;

a plurality of D4-23 gene segments provided by at least 2 differentD4-23 gene; optionally in cis in said genome;

a plurality of D6-13 gene segments provided by at least 2 differentD6-13 gene; optionally in cis in said genome;

a plurality of D3-9 gene segments provided by at least 2 different D3-9gene; optionally in cis in said genome;

a plurality of D4-4 gene segments provided by at least 2 different D4-4gene; optionally in cis in said genome; and

a plurality of D7-27 gene segments provided by at least 2 differentD7-27 gene; optionally in cis in said genome;

wherein the D gene segments are derived from the genome sequence ofdifferent human individuals that are not genetically related over atleast 3 generations.

183. The vertebrate or cell of clause 182, wherein the human individualsare from different human populations.

184. The vertebrate, cell or population of any one of clauses 154 to183, wherein one or more of the D gene segments is a variant of a humangermline D gene segment, wherein the variant gene segment encodes anamino acid sequence that differs by 1, 2 or 3 amino acids from thecorresponding amino acid sequence encoded by the human germline D genesegment, provided in that said amino acid sequence encoded by thevariant does not include a stop codon when said corresponding amino acidsequence does not include a stop codon.

Optionally, the variant and germline D gene segments encode therespective amino acid sequences in reading frame 2 (IMGT numbering). SeeBriney et al 2012.

185. The vertebrate, cell or population of clause 184, wherein saidcorresponding amino acid sequence encoded by the human germline D genesegment is a hydrophilic or hydrophobic sequence (according to J MolBiol. 1997 Jul. 25; 270(4):587-97; Corbett S J et al; Table 2).

186. The vertebrate, cell or population of clause 184 or 185, comprisingsaid variant and said germline human D gene segments; optionally whereinthe variant and germline human D gene segments are cis on the samechromosome.

187. The vertebrate, cell or population of any one of clauses 184 to186, wherein germline human D gene segment is a D2, D3, D5 or D6 familygene segment; optionally a D2-2, D2-15, D3-3, D3-9, D3-10, D3-22, D5-5,D5-18, D6-6, D6-13, D6-19 gene segment.

These D segments are usable in all three reading frames.

Optionally a variant of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of thesehuman germline D gene segments is used.

188. The vertebrate, cell or population of any one of clauses 154 to187, comprising a plurality of D2-2 gene segments, wherein the pluralitycomprises D2-2 gene segments that vary from each other at one or morenucleotide positions corresponding to positions 106,382,687 and106,382,711

on human chromosome 14.

189. The vertebrate, cell or population of clause 188, wherein theplurality comprises a human D2-2 gene segment ((optionally two copiesand/or in homozygous state) comprising a thymine at a positioncorresponding to position 106,382,687 on human chromosome 14; andoptionally no further mutation from the sequence of D2-2ref.

190. The vertebrate, cell or population of clause 188 or 189, whereinthe plurality comprises a human D2-2 gene segment comprising a cytosineat a position corresponding to position 106,382,687 on human chromosome14; and optionally no further mutation from the sequence of D2-2a.

191. The vertebrate, cell or population of any one of clauses 188 to190, wherein the plurality comprises a human D2-2 gene segmentcomprising an adenine at a position corresponding to position106,382,711 on human chromosome 14; and optionally no further mutationfrom the sequence of D2-2b.

192. The vertebrate, cell or population of any one of clauses 188 to191, wherein the plurality comprises a human D2-2 gene segmentcomprising an thymine at a position corresponding to position106,382,711 on human chromosome 14; and optionally no further mutationfrom the sequence of D2-2ref.

193. The vertebrate, cell or population of any one of clauses 154 to192, comprising a plurality of D7-27 gene segments, wherein theplurality comprises D7-27 gene segments that vary from each other at anucleotide position corresponding to position 106,331,767 on humanchromosome 14.

194. The vertebrate, cell or population of clause 193, wherein theplurality comprises a human D7-27 gene segment (optionally two copiesand/or in homozygous state) comprising a cytosine at a positioncorresponding to position 106,331,767 on human chromosome 14; andoptionally no further mutation from the sequence of D7-27ref.

195. The vertebrate, cell or population of clause 193 or 194, whereinthe plurality comprises a human D7-27 gene segment comprising a guanineat a position corresponding to position 106,331,767 on human chromosome14; and optionally no further mutation from the sequence of D7-27a.

196. The vertebrate, cell or population of any one of clauses 154 to195, comprising a plurality of D4-23 gene segments, wherein theplurality comprises D4-23 gene segments that vary from each other at anucleotide position corresponding to position 106,350,740 on humanchromosome 14.

197. The vertebrate, cell or population of clause 196, wherein theplurality comprises a human D4-23 gene segment (optionally two copiesand/or in homozygous state) comprising an adenine at a positioncorresponding to position 106,350,740 on human chromosome 14; andoptionally no further mutation from the sequence of D4-23ref.

198. The vertebrate, cell or population of clause 196 or 197, whereinthe plurality comprises a human D4-23 gene segment (optionally twocopies and/or in homozygous state) comprising an guanine at a positioncorresponding to position 106,350,740 on human chromosome 14; andoptionally no further mutation from the sequence of D4-23a.

199. The vertebrate, cell or population of any one of clauses 154 to197, comprising a plurality of D2-21 gene segments, wherein theplurality comprises D2-21 gene segments that vary from each other at anucleotide position corresponding to position 106,354,418 on humanchromosome 14.

200. The vertebrate, cell or population of clause 199, wherein theplurality comprises a human D2-21 gene segment (optionally two copiesand/or in homozygous state) comprising an adenine at a positioncorresponding to position 106,354,418 on human chromosome 14; andoptionally no further mutation from the sequence of D2-21 ref.

201. The vertebrate, cell or population of clause 199 or 200, whereinthe plurality comprises a human D2-21 gene segment (optionally twocopies and/or in homozygous state) comprising a guanine at a positioncorresponding to position 106,354,418 on human chromosome 14; andoptionally no further mutation from the sequence of D2-21a.

202. The vertebrate, cell or population of any one of clauses 154 to201, comprising a plurality of D3-16 gene segments, wherein theplurality comprises D3-16 gene segments that vary from each other at anucleotide position corresponding to position 106,354,418 on humanchromosome 14.

203. The vertebrate, cell or population of clause 202, wherein theplurality comprises a human D3-16 gene segment (optionally two copiesand/or in homozygous state) comprising a thymine at a positioncorresponding to position 106,361,515 on human chromosome 14; andoptionally no further mutation from the sequence of D3-16ref.

204. The vertebrate, cell or population of clause 202 or 203, whereinthe plurality comprises a human D3-16 gene segment (optionally twocopies and/or in homozygous state) comprising a cytosine at a positioncorresponding to position 106,361,515 on human chromosome 14; andoptionally no further mutation from the sequence of D3-16a.

205. The vertebrate, cell or population of any one of clauses 154 to204, comprising a plurality of D6-13 gene segments, wherein theplurality comprises D6-13 gene segments that vary from each other at anucleotide position corresponding to position 106,367,013 on humanchromosome 14.

206. The vertebrate, cell or population of clause 205, wherein theplurality comprises a human D6-13 gene segment (optionally two copiesand/or in homozygous state) comprising a thymine at a positioncorresponding to position 106,367,013 on human chromosome 14; andoptionally no further mutation from the sequence of D6-13ref.

207. The vertebrate, cell or population of clause 205 or 206, whereinthe plurality comprises a human D6-13 gene segment (optionally twocopies and/or in homozygous state) comprising a cytosine at a positioncorresponding to position 106,367,013 on human chromosome 14; andoptionally no further mutation from the sequence of D6-13a.

208. The vertebrate, cell or population of any one of clauses 154 to207, comprising a plurality of D3-10 gene segments, wherein theplurality comprises D3-10 gene segments that vary from each other at oneor more nucleotide positions corresponding to positions

106.370.370 and

106.370.371

on human chromosome 14.

209. The vertebrate, cell or population of clause 208, wherein theplurality comprises a human D3-10 gene segment (optionally two copiesand/or in homozygous state) comprising a thymine at a positioncorresponding to position 106,370,370 on human chromosome 14; andoptionally no further mutation from the sequence of D3-10ref.

210. The vertebrate, cell or population of clause 208 or 209, whereinthe plurality comprises a human D3-10 gene segment (optionally twocopies and/or in homozygous state) comprising a cytosine at a positioncorresponding to position 106,370,370 on human chromosome 14; andoptionally no further mutation from the sequence of D3-10a.

211. The vertebrate, cell or population of clause 208, 209 or 210wherein the plurality comprises a human D3-10 gene segment (optionallytwo copies and/or in homozygous state) comprising an adenine at aposition corresponding to position 106,370,371 on human chromosome 14;and optionally no further mutation from the sequence of D3-10ref.

212. The vertebrate, cell or population of any one of clauses 208 to211, wherein the plurality comprises a human D3-10 gene segment(optionally two copies and/or in homozygous state) comprising a guanineat a position corresponding to position 106,370,371 on human chromosome14; and optionally no further mutation from the sequence of D3-10b.

213. The vertebrate, cell or population of any one of clauses 154 to212, comprising a plurality of D3-9 gene segments, wherein the pluralitycomprises D3-9 gene segments that vary from each other at a nucleotideposition corresponding to position 106,370,567 on human chromosome 14.

214. The vertebrate, cell or population of clause 213, wherein theplurality comprises a human D3-9 gene segment (optionally two copiesand/or in homozygous state) comprising an adenine at a positioncorresponding to position 106,370,567 on human chromosome 14; andoptionally no further mutation from the sequence of D3-9ref.

215. The vertebrate, cell or population of clause 213 or 214, whereinthe plurality comprises a human D3-9 gene segment (optionally two copiesand/or in homozygous state) comprising a thymine at a positioncorresponding to position 106,370,567 on human chromosome 14; andoptionally no further mutation from the sequence of D3-9a.

216. The vertebrate, cell or population of any one of clauses 154 to215, comprising a plurality of D2-8 gene segments, wherein the pluralitycomprises D2-8 gene segments that vary from each other at one or morenucleotide positions corresponding to positions

106,373,085; 106,373,086 and 106,373,089

on human chromosome 14.

217. The vertebrate, cell or population of clause 216, wherein theplurality comprises a human D2-8 gene segment (optionally two copiesand/or in homozygous state) comprising a cytosine at a positioncorresponding to position 106,373,085 on human chromosome 14.

218. The vertebrate, cell or population of clause 216 or 217, whereinthe plurality comprises a human D2-8 gene segment (optionally two copiesand/or in homozygous state) comprising a thymine at a positioncorresponding to position 106,373,085 on human chromosome 14; andoptionally no further mutation from the sequence of D2-8b.

219. The vertebrate, cell or population of clause 216, 217 or 218wherein the plurality comprises a human D2-8 gene segment (optionallytwo copies and/or in homozygous state) comprising a cytosine at aposition corresponding to position 106,373,086 on human chromosome 14;and

optionally no further mutation from the sequence of D2-8ref.

220. The vertebrate, cell or population of any one of clauses 216 to219, wherein the plurality comprises a human D2-8 gene segmentcomprising a thymine at a position corresponding to position 106,373,086on human chromosome 14; and optionally no further mutation from thesequence of D2-8ref.

221. The vertebrate, cell or population of any one of clauses 154 to220, comprising a plurality of D4-4 gene segments, wherein the pluralitycomprises D4-4 gene segments that vary from each other at one or morenucleotide positions corresponding to positions

106,379,086; and 106,379,089

on human chromosome 14.

222. The vertebrate, cell or population of clause 221, wherein theplurality comprises a D4-4 gene segment (optionally two copies and/or inhomozygous state) comprising a cytosine at a position corresponding toposition 106,379,086 on human chromosome 14; and optionally no furthermutation from the sequence of D4-4ref.

223. The vertebrate, cell or population of clause 221 or 222, whereinthe plurality comprises a human D4-4 gene segment (optionally two copiesand/or in homozygous state) comprising a thymine at a positioncorresponding to position 106,379,086 on human chromosome 14; andoptionally no further mutation from the sequence of D4-4a.

224. The vertebrate, cell or population of clause 221, 222 or 223wherein the plurality comprises a human D4-4 gene segment (optionallytwo copies and/or in homozygous state) comprising a cytosine at aposition corresponding to position 106,379,089 on human chromosome 14;and optionally no further mutation from the sequence of D4-4ref or acytosine at a position corresponding to position 106,379,086 on humanchromosome 14.

225. The vertebrate, cell or population of any one of clauses 221 to224, wherein the plurality comprises a human D4-4 gene segment(optionally two copies and/or in homozygous state) comprising a thymineat a position corresponding to position 106,373,089 on human chromosome14; and optionally no further mutation from the sequence of D4-4a.

226. The vertebrate, cell or population of any one of clauses 154 to225, comprising a plurality of D3-3 gene segments, wherein the pluralitycomprises D3-3 gene segments that vary from each other at one or morenucleotide positions corresponding to positions 106,380,241; and106,380,246 on human chromosome 14.

227. The vertebrate, cell or population of clause 226, wherein theplurality comprises a D3-3 gene segment (optionally two copies and/or inhomozygous state) comprising a thymine at a position corresponding toposition 106,380,241 on human chromosome 14; and optionally no furthermutation from the sequence of D3-3ref.

228. The vertebrate, cell or population of clause 226 or 227, whereinthe plurality comprises a human D3-3 gene segment (optionally two copiesand/or in homozygous state) comprising a cytosine at a positioncorresponding to position 106,380,241 on human chromosome 14; andoptionally no further mutation from the sequence of D3-3a.

229. The vertebrate, cell or population of clause 226, 227 or 228wherein the plurality comprises a human D3-3 gene segment (optionallytwo copies and/or in homozygous state) comprising an adenine at aposition corresponding to position 106,380,246 on human chromosome 14;and optionally no further mutation from the sequence of D3-3ref.

230. The vertebrate, cell or population of any one of clauses 226 to229, wherein the plurality comprises a human D3-3 gene segment(optionally two copies and/or in homozygous state) comprising a thymineat a position corresponding to position 106,380,246 on human chromosome14; and optionally no further mutation from the sequence of D3-3a.

Multiple Human JL Gene Segment Variants

A specific application of this configuration is the provision ofmultiple human JLgene segments (JK and/or JA) as follows (as set out innumbered paragraphs, starting at paragraph number 80).

80. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 3 human JLgene segments of the same type (eg, JK1), wherein atleast two of the human JLgene segments are variants that are notidentical to each other.

81. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 2 different non-endogenous JL gene segments of the same type(eg, JK1) cis at the same Ig locus.

82. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 2 different human JLgene segments of the same type (eg, JK1)trans at the same Ig locus; and optionally a third human JLgene segmentof the same type, wherein the third JL is cis with one of said 2different JL gene segments.

83. A population of non-human vertebrates (eg, mice or rats) comprisinga repertoire of human JL gene segments, wherein the plurality comprisesat least 2 different human JL gene segments of the same type (eg, JK1),a first of said different JL gene segments is provided in the genome ofa first vertebrate of the population, and a second of said differentJLgene segments being provided in the genome of a second vertebrate ofthe population, wherein the genome of the first vertebrate does notcomprise the second JL gene segment.

84. A non-human vertebrate (eg, a mouse or rat) or a non-humanvertebrate cell (eg, an ES cell or a B-cell) having a genome comprisingat least 2 different non-endogenous JL gene segments of the same type(eg, JK1), wherein the JL gene segments are derived from the genomesequence of different human individuals that are not genetically relatedover at least 3 generations.

85. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 3 human JLgene segments of the same type (eg, JK1), wherein at least two of thehuman JL gene segments are variants that are not identical to eachother.

86. A method of enhancing the immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous JLgene segments of the same type (eg, JK1) cis at thesame Ig locus.

87. A method of enhancing the immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differenthuman JLgene segments of the same type (eg, JK1) trans at the same Iglocus; and optionally a third human JLgene segment of the same type,wherein the third JL is cis with one of said 2 different JL genesegments.

88. A method of providing an enhanced human immunoglobulin JL genesegment repertoire, the method comprising providing a population ofnon-human vertebrates (eg, a mouse or rat) comprising a repertoire ofhuman JL gene segments, wherein the method comprises providing at least2 different human JLgene segments of the same type (eg, JK1), wherein afirst of said different JLgene segments is provided in the genome of afirst vertebrate of the population, and a second of said different JLgene segments is provided in the genome of a second vertebrate of thepopulation, wherein the genome of the first vertebrate does not comprisethe second JL gene segment.

89. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 differentnon-endogenous JLgene segments of the same type (eg, JK1), wherein theJL gene segments are derived from the genome sequence of different humanindividuals that are not genetically related over at least 3generations.

90. The vertebrate or cell of paragraph 80, 82 or 84, or the method ofparagraph 85, 82 or 89, wherein at least 2 or 3 of said different genesegments are provided cis at the same Ig locus in said genome.

91. The vertebrate or cell of paragraph 80, 81 or 82, or the method ofparagraph 85, 86 or 87, wherein the JL gene segments are derived fromthe genome sequence of different human individuals that are notgenetically related over at least 3 generations.

92. The vertebrate or cell of paragraph 80, 81 or 82, or the method ofparagraph 85, 86 or 87, wherein the JL gene segments are derived fromthe genome sequence of two or more different human individuals;optionally wherein the different human individuals are from differenthuman populations.

93. The vertebrate, cell or method of paragraph 92, wherein theindividuals are not genetically related.

94. The vertebrate, cell or method of any one of paragraphs 80 to 93,wherein at least one of the different JL segments is a synthetic mutantof a human germline JL gene segment.

95. A method of enhancing the human immunoglobulin gene diversity of anon-human vertebrate (eg, a mouse or rat), the method comprisingproviding the vertebrate with a genome comprising at least 2 human JLgene segments of the same type (eg, JK1), wherein the JL gene segmentsare derived from the genome sequence of different human individuals thatare not genetically related over at least 3 generations; optionallywherein at least 2 or 3 of said different gene segments are provided atthe same IgL locus in said genome.

96. The vertebrate or cell of any one of paragraphs paragraph 80 to 82and 84, wherein the genome comprises a substantially complete functionalrepertoire of human JK and/or Jλ gene segment types supplemented withone, two or more human JK and/or Jλ A gene segments respectively,wherein said substantially complete functional repertoire and thesupplementary gene segments are not found together in the germlinegenome of a human individual.

97. The population of paragraph 83, wherein the population comprises asubstantially complete functional repertoire of human JL gene segmenttypes supplemented with one, two or more human JK and/or Jλ genesegments respectively, wherein said substantially complete functionalrepertoire and the supplementary gene segments are not found together inthe germline genome of a human individual.

98. A non-human vertebrate (eg, a mouse or rat) or a non-human cell (eg,an ES cell or a B-cell) having a genome comprising a substantiallycomplete functional repertoire of human JK and/or Jλ gene segment typessupplemented with one, two or more human JK and/or Jλ gene segmentsrespectively, wherein said substantially complete functional repertoireand the supplementary gene segments are not found together in thegermline genome of a human individual.

99. A population of non-human vertebrates (eg, mice or rats) comprisinga substantially complete functional repertoire of human JK and/or Jλgene segment types supplemented with one, two or more human JK and/or Jλgene segments respectively, wherein said substantially completefunctional repertoire and the supplementary gene segments are not foundtogether in the germline genome of a human individual.

100. A non-human vertebrate or vertebrate cell according to paragraph81, comprising a genome that comprises VL and JL gene repertoirescomprising human gene segments, the JL gene repertoire comprising

a plurality of human JK1 gene segments provided by at least 2 differenthuman JK1 gene segments in cis at the same Ig locus in said genome;

a plurality of human JK2 gene segments provided by at least 2 differenthuman JK1 gene segments in cis at the same Ig locus in said genome;

a plurality of human JK3 gene segments provided by at least 2 differenthuman JK1 gene segments in cis at the same Ig locus in said genome;

a plurality of human JK4 gene segments provided by at least 2 differenthuman JK1 gene segments in cis at the same Ig locus in said genome;

a plurality of human JK5 gene segments provided by at least 2 differenthuman JK1 gene segments in cis at the same Ig locus in said genome;

a plurality of human JA1 gene segments provided by at least 2 differenthuman JA1 gene segments in cis at the same Ig locus in said genome;

a plurality of human JA2 gene segments provided by at least 2 differenthuman JA2 gene segments in cis at the same Ig locus in said genome;

a plurality of human JA3 gene segments provided by at least 2 differenthuman JA3 gene segments in cis at the same Ig locus in said genome;

a plurality of human JA4 gene segments provided by at least 2 differenthuman JA4 gene segments in cis at the same Ig locus in said genome;

a plurality of human JA5 gene segments provided by at least 2 differenthuman JA5 gene segments in cis at the same Ig locus in said genome;

a plurality of human JA6 gene segments provided by at least 2 differenthuman JA6 gene segments in cis at the same Ig locus in said genome; or aplurality of human Jλ7 gene segments provided by at least 2 differenthuman Jλ7 gene segments in cis at the same Ig locus in said genome;

optionally wherein the JLgene segments are derived from the genomesequence of two or more different human individuals.

101. A non-human vertebrate or vertebrate cell (optionally an ES cell orB-cell), according to paragraph 80, comprising a genome that comprisesVL and JL gene repertoires comprising human gene segments, the JL generepertoire comprising

a plurality of human JK1 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JK1 gene segments;

a plurality of human JK2 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JK1 gene segments;

a plurality of human JK3 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JK1 gene segments;

a plurality of human JK4 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JK1 gene segments;

a plurality of human JK5 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JK1 gene segments;

a plurality of human JA1 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JA1 gene segments;

a plurality of human JA2 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JA2 gene segments;

a plurality of human JA3 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JA3 gene segments;

a plurality of human JA4 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JA4 gene segments;

a plurality of human JA5 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JA5 gene segments;

a plurality of human JA6 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human JA6 gene segments; or

a plurality of human Jλ7 gene segments provided by at least 3 (eg, 3, 4,5, 6, or 7) different human Jλ7 gene segments;

optionally wherein the JLgene segments are derived from the genomesequence of two or three different human individuals;

optionally wherein at least 2 or 3 of said different gene segments areprovided in cis at the same Ig locus in said genome.

102. The vertebrate or cell of paragraph 104 or 105, wherein thedifferent human individuals are from different human populations.

103. The vertebrate or cell of any one of paragraphs 104 to 106, whereinthe individuals are not genetically related.

104. The vertebrate or cell of any one of paragraphs 104 to 107, whereinat least one of the different JL segments is a synthetic mutant of ahuman germline JL gene segment.

105. A non-human vertebrate or vertebrate cell (optionally an ES cell orB-cell) according to paragraph 84, comprising a genome comprising humanVL and JL gene repertoires, the JL gene repertoire comprising

a plurality of human JK1 gene segments provided by at least 2 differenthuman JK1 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JK2 gene segments provided by at least 2 differenthuman JK1 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JK3 gene segments provided by at least 2 differenthuman JK1 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JK4 gene segments provided by at least 2 differenthuman JK1 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JK5 gene segments provided by at least 2 differenthuman JK1 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JA1 gene segments provided by at least 2 differenthuman JA1 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JA2 gene segments provided by at least 2 differenthuman JA2 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JA3 gene segments provided by at least 2 differenthuman JA3 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JA4 gene segments provided by at least 2 differenthuman JA4 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JA5 gene segments provided by at least 2 differenthuman JA5 gene segments, optionally in cis at the same Ig locus in saidgenome;

a plurality of human JA6 gene segments provided by at least 2 differenthuman JA6 gene segments, optionally in cis at the same Ig locus in saidgenome; or a plurality of human Jλ7 gene segments provided by at least 2different human Jλ7 gene segments, optionally in cis at the same Iglocus in said genome;

wherein the JL gene segments are derived from the genome sequence ofdifferent human individuals that are not genetically related over atleast 3 generations.

106. The vertebrate or cell of paragraph 109, wherein the humanindividuals are from different human populations.

The skilled person will realise that standard molecular biologytechniques can be used to provide vectors comprising syntheticcombinations of immunoglobulin gene segments (eg, V, D and/or J) for usein the invention, such that the vectors can be used to build atransgenic immunoglobulin locus (eg, using homologous recombinationand/or recombinase mediated cassette exchange as known in the art, eg,see U.S. Pat. No. 7,501,552 (Medarex), U.S. Pat. No. 5,939,598(Abgenix), U.S. Pat. No. 6,130,364 (Abgenix), WO02/066630 (Regeneron),WO2011004192 (Genome Research Limited), WO2009076464, WO2009143472 andWO2010039900 (Ablexis), the disclosures of which are explicitlyincorporated herein. For example, such synthetic combinations of genesegments can be made using standard recombineering techniques in E colito construct BAC vectors harbouring the synthetic combination prior toinsertion in embryonic stem cells using homologous recombination or RMCE(eg, using cre/lox site-specific recombination). Details ofrecombineering can be found at www.genebridges.com and in EP1034260 andEP1204740 the disclosures of which are explicitly incorporated herein.

In one embodiment, it is useful to bias the immune response of thevertebrate (and thus resultant lead antibodies) to a predetermined genesegment, eg, one known to be commonly used in natural human immuneresponses to antigens, such as antigens of infectious disease pathogens.For example, VH1-69 is commonly used to produce antibodies in humansagainst Influenza virus; it is possible, therefore, to include two ormore polymorphic DNA versions of the VH segment VH1-69 in the locus ofthe invention. The examples below illustrate how such a transgenic locuscan be constructed in which diversity is extended by extending theVH1-69 gene segment repertoire based on naturally-occurring VH1-69polymorphic variants.

In one embodiment in any configuration of the invention, the genome hasbeen modified to prevent or reduce the expression of fully-endogenousantibody. Examples of suitable techniques for doing this can be found inPCT/GB2010/051122, U.S. Pat. No. 7,501,552, U.S. Pat. No. 6,673,986,U.S. Pat. No. 6,130,364, WO2009/076464, EP1399559 and U.S. Pat. No.6,586,251, the disclosures of which are incorporated herein byreference. In one embodiment, the non-human vertebrate VDJ region of theendogenous heavy chain immunoglobulin locus, and optionally VJ region ofthe endogenous light chain immunoglobulin loci (lambda and/or kappaloci), have been inactivated. For example, all or part of the non-humanvertebrate VDJ region is inactivated by inversion in the endogenousheavy chain immunoglobulin locus of the mammal, optionally with theinverted region being moved upstream or downstream of the endogenous Iglocus (see, eg, WO2011004192, the disclosure of which is incorporatedherein by reference). For example, all or part of the non-humanvertebrate VJ region is inactivated by inversion in the endogenous kappachain immunoglobulin locus of the mammal, optionally with the invertedregion being moved upstream or downstream of the endogenous Ig locus.For example, all or part of the non-human vertebrate VJ region isinactivated by inversion in the endogenous lambda chain immunoglobulinlocus of the mammal, optionally with the inverted region being movedupstream or downstream of the endogenous Ig locus. In one embodiment theendogenous heavy chain locus is inactivated in this way as is one orboth of the endogenous kappa and lambda loci.

Additionally or alternatively, the vertebrate has been generated in agenetic background which prevents the production of mature host B and Tlymphocytes, optionally a RAG-1-deficient and/or RAG-2 deficientbackground. See U.S. Pat. No. 5,859,301 for techniques of generatingRAG-1 deficient animals.

Thus, in one embodiment of any configuration or aspect of the inventionherein, endogenous heavy and light chain expression has beeninactivated.

In one embodiment each said locus constant region is a heavy chainendogenous non-human vertebrate (optionally host mouse or rat) constantregion.

In one embodiment each said locus constant region is a light chainendogenous non-human vertebrate (optionally host mouse or rat) constantregion.

The invention provides a monoclonal or polyclonal antibody compositionprepared by immunisation of at least one vertebrate (eg, mouse or rat)according to the invention, optionally wherein the antigen is an antigenof an infectious disease pathogen (eg, a bacterial or viral pathogenantigen), optionally wherein the same antigen is used to immunise allthe vertebrates; optionally wherein the antibody or antibodies areIgG-type (eg, IgG1).

The invention also provides a monoclonal or polyclonal antibody mixtureproduced by the method of the invention or a derivative antibody ormixture thereof, eg, where one or more constant region has been changed(eg, replaced with a different constant region such as a human constantregion; or mutated to enhance or ablate Fc effector function). In anaspect of the invention, the monoclonal or polyclonal antibody mixtureis provided for therapy and/or prophylaxis of a disease or condition ina human, eg, for the treatment and/or prevention of an infectiousdisease, wherein optionally wherein each antibody binds an antigen of aninfectious disease pathogen, preferably the same antigen.

In an aspect of the invention, there is provided the use of an isolated,monoclonal or polyclonal antibody according to the invention, or amutant or derivative antibody thereof in the manufacture of a medicamentfor the treatment and/or prevention of a disease or condition in ahuman, eg, an infectious disease, optionally wherein the infectiousdisease is a disease caused by a bacterial or viral pathogen.

An example of a mutant antibody is one that bears up to 15 or 10 aminoacid mutations in its variable regions relative to an isolated antibody(eg, IgG-type, such as IgG1-type, antibody) obtainable or obtained bythe method of the invention. An example of a derivative is one that hasbeen modified to replace a constant region with a different constantregion such as a human constant region; or mutated to enhance or ablateFc effector function.

Examples of infectious diseases are diseases caused or mediated by abacterial or viral pathogen. For example, the infectious disease isselected from the group consisting of a disease caused by a pathogenselected from the group consisting of Haemophilus influenza, E coli,Neisseria meningitidis, a herpes family virus, cytomegalovirus (CMV),HIV and influenza virus.

Tailoring V(D)J Incorporation into Immunoglobin Loci for the Generationof Antibodies Against Infectious Disease

The inventors realised that it would be desirable to provide forvertebrates, cells, methods etc for the production of therapeutic and/orprophylactic antibodies based on natural human immune responses toantigens, such as antigens of infectious disease pathogens. In thisrespect, the literature observes frequently used immunoglobulin genesegments to raise anti-infective responses in humans (Table 9).

In the various configurations, aspects, embodiments and examples above,the invention provides the skilled addressee with the possibility ofchoosing immunoglobulin gene segments in a way that tailors or biasesthe repertoire for application to generating antibodies to treat and/orprevent infectious diseases. The inventors have categorised thefollowing groups of gene segments for use in the invention according tothe desired application of resultant antibodies.

List A:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by a Pathogen

(a) a VL gene segment selected from the group consisting of a VAII genefamily member, VAVII 4A, VAII 2.1, VAVII 4A, a VA1 gene family member, aVA3 gene family member, IGLV1S2, VA3-cML70, IaIh2, IaIvI, Ia3h3, Kv325,a VKI gene family member, KI-15A (KL012), V⁰II family member, a V^(o)IIIfamily member, a VKI gene family member, KI-15A (KL012), V^(o)II A2(optionally the A2a variant), VK A27 (Humkv325) and a gene segment atleast 80% identical thereto.

(b) a VAgene segment selected from a VAII gene family member, VAVII 4A,VAII 2.1, VAVII 4A, a VA1 gene family member, a VA3 gene family member,IGLV1S2, VA3-cML70, IaIh2, IaIvI, Ia3h3 and a gene segment at least 80%identical thereto.

(c) a VK gene segment selected from Kv325, a VKI gene family member,KI-15A (KL012), V^(o)II family member, a VKIII family member, a VKI genefamily member, KI-15A (KL012), V^(o)II A2 (optionally the A2a variant),VK A27 (Humkv325) and a gene segment at least 80% identical thereto.

(d) a VH gene segment a VHIII gene family member (optionally, a VHIIIaor VHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3-15),VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11,VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2,Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%identical thereto.

(e) a Jλ gene segment selected from JA2, JA3 and a gene segment at least80% identical thereto.

a D gene segment selected from Dk1, Dxp>>1, Dn4r, D2r and a gene segmentat least 80% identical thereto.

List A1:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by a Pathogen

(a) a VAgene segment selected from a VAII gene family member, VAVII 4A,VAII 2.1, VAVII 4A and a gene segment at least 80% identical thereto.

(b) a VK gene segment selected from a VKI gene family member, KI-15A(KL012), V^(o)II family member, a VKIII family member, a VKI gene familymember, KI-15A (KL012), V^(o)II A2 (optionally the A2a variant), VK A27(Humkv325) and a gene segment at least 80% identical thereto.

(c) a VH gene segment a VH3 gene family member (optionally, a VHIIIa orVHIIIb family member), VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21,LSG6.1, LSG12.1, DP77 (V3-21), VH H11 and a gene segment at least 80%identical thereto.

(d) a Jλ gene segment selected from JA2, JA3 and a gene segment at least80% identical thereto.

(e) a JH gene segment selected from JH2, JH3, JH4 and a gene segment atleast 80% identical thereto.

List A1.1:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by H Influenza

(a) a VAgene segment selected from a VAII gene family member, VAVII 4A,VAII 2.1, VAVII 4A and a gene segment at least 80% identical thereto.

(b) a VK gene segment selected from a V^(o)II family member, a VKIIIfamily member, a VKI gene family member, KI-15A (KL012), V^(o)II A2(optionally the A2a variant), V^(o) A27 (Humkv325) and a gene segment atleast 80% identical thereto.

(c) a VH gene segment a VH3 gene family member (optionally, a VHIIIbfamily member), VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1,LSG12.1, DP77 (V3-21) and a gene segment at least 80% identical thereto.

(d) a Jλ gene segment selected from JA2, JA3 and a gene segment at least80% identical thereto.

List A1.2:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by E Coli or Neisseria meningitidis

(a) a VH gene segment a VH3 gene family member (optionally a VHIIIa orVHIIIb member), VHIII 9.1 (VH3-15), VH H11, VHIII VH26 (VH3-23) a genesegment at least 80% identical thereto, eg, VHIII 9.1 ©JH3; or VH H11JH4; or VHIII VH26 © JH2.

(b) a VK gene segment selected from a VKI gene family member, KI-15A(KL012) and a gene segment at least 80% identical thereto.

(c) a VAgene segment selected from a VAII gene family member, VAII 2.1and a gene segment at least 80% identical thereto.

(d) a JH gene segment selected from JH2, JH3, JH4 and a gene segment atleast 80% identical thereto.

A2:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by a Viral Pathogen

(a) a VH gene segment selected from a VHIII gene family member, a VHIVgene family member, VHIII-VH26 (VH3-23), VH1GRR, ha3h2, VHI-ha1c1,VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251,VH1-69 and a gene segment at least 80% identical thereto.

(b) a VA gene segment selected from a VA1 gene family member, a VA3 genefamily member, IGLV1S2, VA3-cML70, IaIh2, IaIvI, Ia3h3 and a genesegment at least 80% identical thereto.

(c) a Vk gene segment selected from Kv325 and a gene segment at least80% identical thereto.

(d) a JH gene segment selected from JH3, JH5, JH6 and a gene segment atleast 80% identical thereto.

(e) a D gene segment selected from Dk1, Dxp>>1, Dn4r, D2r and a genesegment at least 80% identical thereto.

a Jλ gene segment selected from JA2, JA3 and a gene segment at least 80%identical thereto.

A2.1:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by Herpes Virus Family (eq, VZV or HSV)

(a) a VH gene segment selected from a VHIII gene family member, a VHIVgene family member, VHIII-VH26 (VH3-23), VH1GRR, ha3h2, VHI-ha1c1,VHIII-VH2-1, VH4.18, ha4h3, and a gene segment at least 80% identicalthereto.

(b) a VA gene segment selected from a VA1 gene family member, a VA3 genefamily member, IGLV1S2, VA3-cML70, IaIh2, IaIvI, Ia3h3 and a genesegment at least 80% identical thereto.

(c) a JH gene segment selected from JH3, JH5, JH6 and a gene segment atleast 80% identical thereto.

(d) a D gene segment selected from Dk1, Dxp>>1, Dn4r, D2r and a genesegment at least 80% identical thereto.

(e) a Jλ gene segment selected from JA2, JA3 and a gene segment at least80% identical thereto.

A2.2:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by CMV

(a) a VH gene segment selected from Hv1051 and a gene segment at least80% identical thereto.

(b) a Vk gene segment selected from Kv325 and a gene segment at least80% identical thereto. A2.3:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by HIV

(a) a VH gene segment selected from 71-2, Hv1f10, VH4.11, 71-4, VH251,VH1-69 and a gene segment at least 80% identical thereto.

A2.4:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by Influenza Virus

(a) a VH gene segment selected from VH1-69 and a gene segment at least80% identical thereto.

Thus,

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease, one or more V, D and/or or all Jgene segments used in any configuration, aspect, method, example orembodiment of the invention can be selected from List A1. Thus, forexample in (a) of the first configuration of the invention, the recitedheavy chain V gene segment is selected from the VH gene segments in ListA, optionally with a D in that list.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by a bacterialpathogen, one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A1.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by a viralpathogen, one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A2.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by H influenza,one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A1.1.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by E Coli orNeisseria meningitidis, one or more or all V, D and/or J gene segmentsused in any configuration, aspect, method, example or embodiment of theinvention can be selected from List A1.2.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by Herpes VirusFamily (eg, VZV or HSV), one or more or all V, D and/or J gene segmentsused in any configuration, aspect, method, example or embodiment of theinvention can be selected from List A2.1.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by CMV, one ormore or all V, D and/or J gene segments used in any configuration,aspect, method, example or embodiment of the invention can be selectedfrom List A2.2.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by HIV, one ormore or all V, D and/or J gene segments used in any configuration,aspect, method, example or embodiment of the invention can be selectedfrom List A2.3.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by InfluenzaVirus, one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A2.4.

Optionally each VH segment in the locus of the invention is selectedfrom List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Optionally each VL segment in the locus of the invention is selectedfrom List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4

Optionally each D segment in the locus of the invention is selected fromList A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Optionally each JL segment in the locus of the invention is selectedfrom List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Antibodies for Therapy & Prophylaxis of Patients of Specific Ancestry

The inventors, having undertaken the extensive Bioinformatics analysisexercise described herein, realised that the output of that analysis hasmade it possible to identify specific gene segments that are useful toproduce antibody- and VH domain-based drugs that are tailoredspecifically to a patient's ancestry (ie, genotype). That is, antibodiescan be selected on the basis that they are made in vivo in a transgenicnon-human vertebrate (eg, mouse or rat with transgenic IgH loci) andparticularly derived from gene segments that are relatively prevalent inmembers of the patient's population, ie, from individuals of the samehuman ancestry. Since variant distributions differ across differentpopulations (see Table 13), this presumably reflects the effects ofevolution, adaptation and conservation of useful variant gene types inthose populations. Thus, by tailoring the antibody-based drugs accordingto the invention, it is possible to match the drug to the populationgene biases, thus with the aim of making better drugs for that specificpopulation of humans. Better can, for example, mean more efficacious,better neutralising, higher target antigen affinity, less immunogenic,less patient reactions to the drug etc. This can be determinedempirically, as is standard in drug research and development processes.

Thus, the Invention Provides the Following Embodiments (Numbered fromClause 345 Onwards):—

345. An isolated antibody for administration to a Chinese patient, theantibody comprising a human heavy chain, the heavy chain comprising avariable domain that is specific for an antigen and a constant region,wherein the constant region is a human constant region selected from aconstant region (eg, an IGHG constant region) in Table 13 found in aChinese population and with a cumulative frequency of at least 1 or 5%;and wherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat);and/or (ii) the variable domain comprises non-human vertebrate (eg,mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouseor rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In another embodiment, the invention provides

An isolated antibody for administration to a Chinese patient, theantibody comprising a human heavy chain, the heavy chain comprising avariable domain that is specific for an antigen and a constant region,wherein the constant region is a human constant region selected from aconstant region (eg, an IGHG constant region) present in a Chinesepopulation with a cumulative frequency of at least 5%; and wherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat);and/or (ii) the variable domain comprises non-human vertebrate (eg,mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouseor rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In an example, the constant region is found in the 1000 Genomesdatabase. In an example, the constant region is found in Table 13.

346. The antibody of clause 345 wherein the constant region is a IGHG1a,IGHG2a, IGHG3a, IGHG3b or IGHG4a constant region.

347. The antibody of clause 345 or 346, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the VH gene segment beingselected from a VH in Table 13 found in a Chinese population and with acumulative frequency of at least 5%.

In another embodiment, the invention provides

The antibody of clause 345 or 346, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the VH gene segment beingselected from a VH present in a Chinese population with a cumulativefrequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

348. The antibody of clause 345, 346 or 347, wherein the variable domainis derived from the recombination of a human VH gene segment with ahuman D gene segment and a human JH gene segment, the D gene segmentbeing selected from a D in Table 13 found in a Chinese population andwith a cumulative frequency of at least 5%.

In another embodiment, the invention provides

The antibody of clause 345, 346 or 347, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the D gene segment beingselected from a D present in a Chinese population with a cumulativefrequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

349. The antibody of clause 345, 346, 347 or 348 wherein the variabledomain is derived from the recombination of a human VH gene segment witha human D gene segment and a human JH gene segment, the JH gene segmentbeing selected from a JH in Table 13 found in a Chinese population andwith a cumulative frequency of at least 5%.

In another embodiment, the invention provides

The antibody of clause 345, 346, 347 or 348 wherein the variable domainis derived from the recombination of a human VH gene segment with ahuman D gene segment and a human JH gene segment, the JH gene segmentbeing selected from a JH present in a Chinese population with acumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

350. An isolated VH domain identical to a variable domain as recited inany one of clauses 347 to 349, optionally fused at its C-terminus to apolypeptide (eg, an antibody Fc).

In an embodiment, there is provided an isolated VH domain identical to avariable domain as recited in any one of clauses 347 to 349 which ispart of a conjugate, conjugated with a label (eg, for imaging in thepatient) or a toxin (eg, a radioactive toxic payload, such as for cancertreatment in the patient) or a half-life-extending moiety (eg, PEG ofhuman serum albumin).

351. A pharmaceutical composition comprising the antibody or variabledomain of any one of clauses 345 to 350 together with apharmaceutically-acceptable excipient, diluent or a medicament (eg, afurther antigen-specific variable domain, antibody chain or antibody).

352. An isolated antibody for administration to a Chinese patient, theantibody comprising a human heavy chain, the heavy chain comprising avariable domain that is specific for an antigen and a constant region,wherein the variable domain is derived from the recombination of a humanVH gene segment with a human D gene segment and a human JH gene segment,the VH gene segment being selected from a VH in Table 13 found in aChinese population and with a cumulative frequency of at least 5%; andwherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat);and/or (ii) the variable domain comprises non-human vertebrate (eg,mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouseor rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In another embodiment, the invention provides

An isolated antibody for administration to a Chinese patient, theantibody comprising a human heavy chain, the heavy chain comprising avariable domain that is specific for an antigen and a constant region,wherein the variable domain is derived from the recombination of a humanVH gene segment with a human D gene segment and a human JH gene segment,the VH gene segment being selected from a VH present in a Chinesepopulation with a cumulative frequency of at least 5%; and wherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat);and/or (ii) the variable domain comprises non-human vertebrate (eg,mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouseor rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

353. The antibody of clause 352, wherein the variable domain is derivedfrom the recombination of a human VH gene segment with a human D genesegment and a human JH gene segment, the D gene segment being selectedfrom a D in Table 13 found in a Chinese population and with a cumulativefrequency of at least 5%.

In another embodiment, the invention provides

The antibody of clause 352, wherein the variable domain is derived fromthe recombination of a human VH gene segment with a human D gene segmentand a human JH gene segment, the D gene segment being selected from a Dpresent in a Chinese population with a cumulative frequency of at least5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

354. The antibody of clause 352 or 353, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the JH gene segment beingselected from a JH in Table 13 found in a Chinese population and with acumulative frequency of at least 5%.

In another embodiment, the invention provides

The antibody of clause 352 or 353, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the JH gene segment beingselected from a JH present in a Chinese population with a cumulativefrequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

355. An isolated VH domain identical to a variable domain as recited inany one of clauses 352 to 354, optionally fused at its C-terminus to apolypeptide (eg, an antibody Fc).

In an embodiment, there is provided a VH domain identical to a variabledomain as recited in any one of clauses 352 to 354 which is part of aconjugate, conjugated with a label (eg, for imaging in the patient) or atoxin (eg, a radioactive toxic payload, such as for cancer treatment inthe patient) or a half-life-extending moiety (eg, PEG of human serumalbumin).

356. A pharmaceutical composition comprising the antibody or variabledomain of any one of clauses 352 to 355 together with apharmaceutically-acceptable excipient, diluent or a medicament (eg, afurther antigen-specific variable domain, antibody chain or antibody).

357. An antibody heavy chain or VH domain (eg, provided as part of anantibody) for therapy and/or prophylaxis of a disease or medicalcondition in a Chinese patient, wherein the heavy chain is a heavy chainproduced by the following steps (or is a copy of such a heavy chain):—

(a) Selection of an antigen-specific antibody heavy chain or VH domainfrom a non-human vertebrate (eg, a mouse or a rat), wherein the heavychain or VH domain is derived from the recombination of a human VH genesegment with a human D gene segment and a human JH gene segment, the VHgene segment being selected from a VH in Table 13 found in a Chinesepopulation and with a cumulative frequency of at least 5%;

(b) Optional humanisation of the heavy chain by combining the variabledomain of the heavy chain with a human constant region; or optionalhumanisation of the selected VH domain by combining with a humanconstant region.

In another embodiment, the invention provides

An antibody heavy chain or VH domain (eg, provided as part of anantibody) for therapy and/or prophylaxis of a disease or medicalcondition in a Chinese patient, wherein the heavy chain is a heavy chainproduced by the following steps (or is a copy of such a heavy chain):—

(a) Selection of an antigen-specific antibody heavy chain or VH domainfrom a non-human vertebrate (eg, a mouse or a rat), wherein the heavychain or VH domain is derived from the recombination of a human VH genesegment with a human D gene segment and a human JH gene segment, the VHgene segment being selected from a VH present in a Chinese populationwith a cumulative frequency of at least 5%;

(b) Optional humanisation of the heavy chain by combining the variabledomain of the heavy chain with a human constant region; or optionalhumanisation of the selected VH domain by combining with a humanconstant region.

In an example, the VH gene segment is found in the 1000 Genomesdatabase. In an example, the gene segment is found in Table 13.

358. The antibody heavy chain or VH domain of clause 357, wherein thehuman constant region is as recited in clause 345 or 346.

359. An antibody heavy chain or VH domain as recited in clause 357 or358 for use in a medicament for therapy and/or prophylaxis of a diseaseor medical condition in a Chinese patient.

360. A method of treating and/or preventing a disease or medicalcondition in a Chinese patient, the method comprising administering tothe patient a therapeutically or prophylactically-effective amount ofthe antibody heavy chain or VH domain as recited in clause 357 or 358.

361. An isolated antibody for administration to a patient of European,East Asian, West African, South Asian or Americas ancestry, the antibodycomprising a human heavy chain, the heavy chain comprising a variabledomain that is specific for an antigen and a constant region, whereinthe constant region is a human constant region selected from a constantregion (eg, an IGHG constant region) in Table 13 found in a populationof European, East Asian, West African, South Asian or Americas ancestryrespectively and with a cumulative frequency of at least 1 or 5%; andwherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat); or(ii) the variable domain comprises non-human vertebrate (eg, mouse orrat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat)terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In another embodiment, the invention provides

An isolated antibody for administration to a patient of European, EastAsian, West African, South Asian or Americas ancestry, the antibodycomprising a human heavy chain, the heavy chain comprising a variabledomain that is specific for an antigen and a constant region, whereinthe constant region is a human constant region selected from a constantregion (eg, an IGHG constant region) present in a population ofEuropean, East Asian, West African, South Asian or Americas ancestryrespectively with a cumulative frequency of at least 1 or 5%; andwherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat); or(ii) the variable domain comprises non-human vertebrate (eg, mouse orrat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat)terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In an example, the constant region is found in the 1000 Genomesdatabase. In an example, the constant region is found in Table 13.

362. The antibody of clause 361 wherein the constant region is a IGHG1a,IGHG2a, IGHG3a, IGHG3b or IGHG4a constant region and the patient is ofEuropean ancestry.

363. The antibody of clause 361 or 362, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the VH gene segment beingselected from a VH in Table 13 found in said population and with acumulative frequency of at least 1 or 5%.

In another embodiment, the invention provides

The antibody of clause 361 or 362, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the VH gene segment beingselected from a VH present in a Chinese population with a cumulativefrequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

364. The antibody of clause 361, 362 or 363, wherein the variable domainis derived from the recombination of a human VH gene segment with ahuman D gene segment and a human JH gene segment, the D gene segmentbeing selected from a D in Table 13 found in said population and with acumulative frequency of at least 1 or 5%.

In another embodiment, the invention provides

The antibody of clause 361, 362 or 363, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the D gene segment beingselected from a D present in a Chinese population with a cumulativefrequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

365. The antibody of clause 361, 362, 363 or 364 wherein the variabledomain is derived from the recombination of a human VH gene segment witha human D gene segment and a human JH gene segment, the JH gene segmentbeing selected from a JH in Table 13 found in said population and with acumulative frequency of at least 1 or 5%.

In another embodiment, the invention provides

The antibody of clause 361, 362, 363 or 364 wherein the variable domainis derived from the recombination of a human VH gene segment with ahuman D gene segment and a human JH gene segment, the JH gene segmentbeing selected from a JH present in a Chinese population with acumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

366. An isolated VH domain identical to a variable domain as recited inany one of clauses 363 to 365, optionally fused at its C-terminus to apolypeptide (eg, an antibody Fc).

367. A pharmaceutical composition comprising the antibody or variabledomain of any one of clauses 361 to 366 together with apharmaceutically-acceptable excipient, diluent or a medicament (eg, afurther antigen-specific variable domain, antibody chain or antibody).

368. An isolated antibody for administration to a patient of European,East Asian, West African or Americas ancestry, the antibody comprising ahuman heavy chain, the heavy chain comprising a variable domain that isspecific for an antigen and a constant region, wherein the variabledomain is derived from the recombination of a human VH gene segment witha human D gene segment and a human JH gene segment, the VH gene segmentbeing selected from a VH in Table 13 found in a population of European,East Asian, West African, South Asian or Americas ancestry respectivelyand with a cumulative frequency of at least 1 or 5%; and wherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat); or(ii) the variable domain comprises non-human vertebrate (eg, mouse orrat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat)terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In another embodiment the invention provides:—

An isolated antibody for administration to a patient of European, EastAsian, West African or Americas ancestry, the antibody comprising ahuman heavy chain, the heavy chain comprising a variable domain that isspecific for an antigen and a constant region, wherein the variabledomain is derived from the recombination of a human VH gene segment witha human D gene segment and a human JH gene segment, the VH gene segmentbeing selected from a VH present in a population of European, EastAsian, West African, South Asian or Americas ancestry respectively witha cumulative frequency of at least 1 or 5%; and wherein

(i) the variable domain is derived from the recombination of said humangene segments in a non-human vertebrate (eg, in a mouse or a rat); or(ii) the variable domain comprises non-human vertebrate (eg, mouse orrat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat)terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In an example, the VH gene segment is found in the 1000 Genomesdatabase. In an example, the gene segment is found in Table 13.

369. The antibody of clause 368, wherein the variable domain is derivedfrom the recombination of a human VH gene segment with a human D genesegment and a human JH gene segment, the D gene segment being selectedfrom a D in Table 13 found in said population and with a cumulativefrequency of at least 1 or 5%.

In another example there is provided

The antibody of clause 368, wherein the variable domain is derived fromthe recombination of a human VH gene segment with a human D gene segmentand a human JH gene segment, the D gene segment being selected from a Dpresent in said population with a cumulative frequency of at least 1 or5%.

In an example, the D gene segment is found in the 1000 Genomes database.In an example, the gene segment is found in Table 13.

370. The antibody of clause 368 or 369, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the JH gene segment beingselected from a JH in Table 13 found in said population and with acumulative frequency of at least 1 or 5%.

In another example there is provided

The antibody of clause 368 or 369, wherein the variable domain isderived from the recombination of a human VH gene segment with a human Dgene segment and a human JH gene segment, the JH gene segment beingselected from a JH present in said population and with a cumulativefrequency of at least 1 or 5%.

In an example, the JH gene segment is found in the 1000 Genomesdatabase. In an example, the gene segment is found in Table 13.

371. An isolated VH domain identical to a variable domain as recited inany one of clauses 368 to 370, optionally fused at its C-terminus to apolypeptide (eg, an antibody Fc).

372. A pharmaceutical composition comprising the antibody or variabledomain of any one of clauses 368 to 371 together with apharmaceutically-acceptable excipient, diluent or a medicament (eg, afurther antigen-specific variable domain, antibody chain or antibody).

373. An antibody heavy chain or VH domain (eg, provided as part of anantibody) for therapy and/or prophylaxis of a disease or medicalcondition in a patient of European, East Asian, West African, SouthAsian or Americas ancestry, wherein the heavy chain is a heavy chainproduced by the following steps (or is a copy of such a heavy chain):—

(a) Selection of an antigen-specific antibody heavy chain or VH domainfrom a non-human vertebrate (eg, a mouse or a rat), wherein the heavychain or VH domain is derived from the recombination of a human VH genesegment with a human D gene segment and a human JH gene segment, the VHgene segment being selected from a VH in Table 13 found in saidpopulation and with a cumulative frequency of at least 1 or 5%;

(b) Optional humanisation of the heavy chain by combining the variabledomain of the heavy chain with a human constant region; or optionalhumanisation of the selected VH domain by combining with a humanconstant region.

In another embodiment, there is provided:—

An antibody heavy chain or VH domain (eg, provided as part of anantibody) for therapy and/or prophylaxis of a disease or medicalcondition in a patient of European, East Asian, West African, SouthAsian or Americas ancestry, wherein the heavy chain is a heavy chainproduced by the following steps (or is a copy of such a heavy chain):—

(a) Selection of an antigen-specific antibody heavy chain or VH domainfrom a non-human vertebrate (eg, a mouse or a rat), wherein the heavychain or VH domain is derived from the recombination of a human VH genesegment with a human D gene segment and a human JH gene segment, the VHgene segment being selected from a VH present in said population with acumulative frequency of at least 1 or 5%;

(b) Optional humanisation of the heavy chain by combining the variabledomain of the heavy chain with a human constant region; or optionalhumanisation of the selected VH domain by combining with a humanconstant region.

In an example, the VH gene segment is found in the 1000 Genomesdatabase. In an example, the gene segment is found in Table 13.

374. The antibody heavy chain or VH domain of clause 373, wherein thehuman constant region is as recited in clause 361 or 362.

375. An antibody heavy chain or VH domain as recited in clause 373 or374 for use in a medicament for therapy and/or prophylaxis of a diseaseor medical condition in a patient of said ancestry.

376. A method of treating and/or preventing a disease or medicalcondition in a patient of European, East Asian, West African, SouthAsian or Americas ancestry, the method comprising administering to thepatient a therapeutically or prophylactically-effective amount of theantibody heavy chain or VH domain as recited in clause 373 or 374.

In embodiments herein, a Chinese patient can be a Han Chinese patient.

In embodiments herein, a patient of European ancestry can be a patientof Northern or Western European ancestry, Italian ancestry, British orScottish ancestry, Finnish ancestry or Iberian ancestry.

In embodiments herein, a patient of East Asian ancestry can be a patientof Han Chinese ancestry, Japanese ancestry Chinese Dai ancestry,Vietnamese ancestry or Kinh ancestry.

In embodiments herein, a patient of West African ancestry can be apatient of Yoruba ancestry, Luhya ancestry, Gambian ancestry or Malawianancestry.

In embodiments herein, a patient of Americas ancestry can be a patientof African American ancestry, African Caribbean ancestry, Mexicanancestry, Puerto Rican ancestry, Colombian ancestry or Peruvianancestry.

In embodiments herein, a patient of South Asian ancestry can be apatient of Ahom ancestry, Kayadtha ancestry, Reddy ancestry, Marathaancestry, or Punjabi ancestry.

In an example of any aspect, the cumulative frequency is at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90 or 95%.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. The use of the word “a” or“an” when used in conjunction with the term “comprising” in the claimsand/or the specification may mean “one,” but it is also consistent withthe meaning of “one or more,” “at least one,” and “one or more thanone.” The use of the term or in the claims is used to mean “and/or”unless explicitly indicated to refer to alternatives only or thealternatives are mutually exclusive, although the disclosure supports adefinition that refers to only alternatives and “and/or.” Throughoutthis application, the term “about” is used to indicate that a valueincludes the inherent variation of error for the device, the methodbeing employed to determine the value, or the variation that existsamong the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof is intended to include atleast one of: A, B, C, AB, AC, BC, or ABC, and if order is important ina particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or

methods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. All such similar substitutes and modifications apparentto those skilled in the art are deemed to be within the spirit, scopeand concept of the invention as defined by the appended claims.

The present invention is described in more detail in the following nonlimiting prophetic Examples.

EXAMPLES Example 1 Recombineered BAC Vectors to Add PolymorphicV-Regions to the Mouse Genome

FIG. 1 through 3 depict recombineering methods (see references above)that can be used to introduce polymorphic V-gene regions into genomicDNA. In one embodiment, a genomic fragment from the human heavy chainregion is inserted into a bacterial artificial chromosome (BAC) vectorby standard techniques. Preferably, such a BAC, which can range in sizefrom 20-kb to 200-kb or more, can be isolated from libraries of BACs bystandard techniques including sequence searches of commerciallyavailable libraries or by hybridization to bacterial colonies containingBACs to identify those with a BAC of interest.

A BAC is chosen that has several VH gene segments; in FIG. 1, these aregenerically identified as VH[a] through VH[z] for example. One skilledin the art will readily identify appropriate genomic fragments, forexample, an approximately 120-kb fragment from human VH5-78 throughVH1-68 which includes 5 endogenous active VH gene segments and 7 VHpsuedogenes. Using recombineering techniques, the endogenous VH genesegments can be replaced by polymorphic VH or VL gene segments. In thisexample, two steps are required. The first step replaces the V-regioncoding exon of an endogenous VH gene segment with a positive-negativeselection operon, in this example, an operon encoding an ampicillinresistance gene (Amp) and a streptomycin-sensitizing ribosomal protein(rpsL). Certain strains of bacteria can be selected for the absence ofthe rpsL gene by resistance to streptomycin. Short stretches of DNAhomologous to sequences flanking the endogenous VH gene exon are placed5′ and 3′ of the rpsL-Amp operon. In the presence of appropriaterecombination factors per standard recombineering techniques (seereferences above) recombination between the operon fragment and the BACwill result in replacement of the endogenous VH gene exon with theoperon (FIG. 1a ) which are selected by resistance to ampicillin. Thesecond step uses the same homologous sequences in order to replace theinserted operon with a desired polymorphic VH gene segment. In thisexample, a human VH1-69 gene is inserted (FIGS. 1b and 1c ). Inparticular the *02 variant of VH1-69 is used [ref IMGT and FIG. 5].Successful integrations of the polymorphic VH gene segment are selectedin bacteria that become resistant to streptomycin due to the loss of theoperon, specifically the rpsL portion.

In this example, the two step process as described can be repeated foreach of the endogenous VH gene segments or for as many endogenous genesegments that one wishes to replace with polymorphic V gene segments(FIG. 1d ).

As is apparent, any polymorphic V gene segment can be inserted in thismanner and any endogenous V gene segment can act as a target, includingpseudogenes. V gene segments in each of the heavy chain and two lightchain loci can be replaced using this technique with appropriate genomicfragments available as BAC inserts.

FIG. 2 depicts another method for creating a genomic fragment encodingpolymorphic V gene segments. In this example, polymorphic V genesegments are inserted into a region of genomic DNA devoid of othergenes, control elements or other functions. Such ‘desert’ regions can beselected based on sequence analysis and corresponding DNA fragmentscloned into BACs or identified in existing BAC libraries. Starting withsuch a genomic fragment, recombineering techniques can be used to insertpolymorphic V gene segments at intervals of, for example, 10-kb. In thisexample, a 150-kb genomic fragment might accommodate insertion of up to15 polymorphic V gene segments. Insertion of the segments is a two-stepprocess. The first recombineering step inserts the rpsL-Amp operon at aspecific site. Sequences homologous to a specific site are used to flankthe operon. These are used by the recombineering system to insert theelement specifically into the BAC genomic fragment and positive eventsare selected by resistance to ampicillin (FIG. 2a ). The second stepreplaces the operon in the genomic fragment with a polymorphic V genesegment by a similar recombineering step using the same sequencehomology (FIG. 2b ). In this example, both exons and promoter element ofa polymorphic VH gene segment are inserted, resulting in replacement ofthe rpsL-Amp operon and therefore resistance to streptomycin (FIG. 2c ).

The two step technique for inserting polymorphic V gene segments into aspecific site on the genomic fragment can be repeated multiple timesresulting in a BAC genomic fragment with several polymorphic genesegments, including their promoter elements. It is apparent that theexamples shown in FIGS. 1 and 2 can be combined wherein the techniquefor insertion can be used to add extra polymorphic V gene segments to aBAC genomic fragment as depicted in FIG. 1. One might choose to addthese extra segments to an IG genomic fragment since such a fragmentwould be more amenable to proper IG gene expression once inserted into anon-human mammal's genome. It is known that a genomic fragment can haveelements such as enhancers or elements that contribute to certainchromatin conformations, both important in wild-type gene expression.

FIG. 3 depicts an additional method to create genomic fragments withpolymorphic V gene segments. This method depends upon the efficiencywith which short (around 50 to 150 bases, preferably 100 bases) singlestranded DNA fragments recombine with a homologous sequence usingrecombineering (Nat Rev Genet. 2001 October; 2(10):769-79;Recombineering: a powerful new tool for mouse functional genomics;Copeland N G, Jenkins N A, Court D L). The recombinases used inrecombineering preferentially bind and use such short single-strandedfragments of DNA as a substrate for initiating homologous recombination.The efficiency can be as high as 10-2, that is, a positive event can befound in approximately 100 randomly picked (not selected) clonesresulting from recombineering. A positive event in this exampleoccurring when one or more single nucleotide changes introduced into thesingle-stranded fragment get transferred to the BAC insert containing Vgene segments and surrounding genomic DNA, said nucleotide change orchanges occurring at a homologous sequence on the BAC.

Polymorphic V gene segments can differ from endogenous V gene segmentsby only 1 or 2, or up to 10 or 15 nucleotide changes, for example. Anexample of such nucleotide polymorphisms are depicted in FIG. 5. Shortsingle stranded regions that encompass the polymorphic nucleotidechanges can be chemically synthesized using standard techniques. Theresulting single stranded DNA fragments are introduced into bacteria andvia recombineering techniques approximately 1 in 100 BAC fragments willhave incorporated the polymorphic nucleotides via homologousincorporation of the single stranded fragment (FIG. 3a ). BACs with thedesired nucleotide change can be identified by screening for exampleseveral hundred individual clones by polymerase chain reaction (PCR)amplification and sequencing, both by standard techniques. In theexample, two nucleotide changes will convert a VH1-69*01 gene segmentinto a VH1-69*02 gene segment (FIG. 3b ).

It is clear that this process can be repeated for multiple endogenous Vgene segments contained on a single BAC genomic fragment. In addition,the techniques depicted in FIG. 2 can be used to add additionalpolymorphic V gene segments by insertion into regions between existing Vgene segments. As would be evident to one skilled in the art, acombination of these techniques can be used to create numerousvariations of both polymorphic and endogenous human V gene segments. Andit would be evident that several different genomic fragments withengineered polymorphic V gene segments and endogenous human V genesegments can be combined to create even more variations.

Example 2 Adding Polymorphic V-Regions to the Genome Using SRMCE ofModified BACs

Modified BACs with polymorphic V gene segments created using the methodsdescribed in Example 1 can be used to alter the genome of non-humanmammals. These alterations can result in an intact IG locus in whichnormal immunoglobin region recombination results in VDJ or VJcombinations which includes the human V gene segments. An example of howsuch an animal can be created is by altering the genome of, for example,mouse embryonic stem (ES) cells using the strategy outlined in FIG. 4.

One technique to integrate modified BACs with polymorphic V genesegments into a genome is sequential recombinase mediated cassetteexchange (SRMCE). The technique is described in WO2011004192 (GenomeResearch Limited), which is incorporated here in its entirety byreference.

SRMCE provides for a locus modified with a ‘landing pad’ inserted at aspecific location. This insertion can either be de novo via homologousrecombination or as a consequence of a previous BAC insertion. In thisexample, the landing pad is inserted in the mouse IGH locus between themost 3′ J gene segment and the Ĉ gene segment and a previous BACinsertion via SRMCE techniques have resulted in the addition of 5 humanV gene segments and 2 V region pseudogenes. The landing pad has elementsas shown in FIG. 4 that will allow the selection of correct insertion ofa second targeting BAC fragment. The specificity of this insertion isprovided by cre recombinase-mediated exchange between permissive loxsites. A lox site is permissive for recombination only with a compatiblelox site. In this example, the loxP site will only recombine with loxPand lox2272 will only recombine with lox2272. This providesdirectionality to the insertion of the BAC fragment as depicted in FIGS.4b and 4 c.

ES cell clones with correct insertions are selected from a pool ofclones without insertions or with non-productive insertions byresistance to puromycin. Resistance to puromycin results from thejuxtaposition of an active promoter element, PGK, with the puroTK codingregion. Correct insertions are verified by standard techniques includingPCR of junctions, PCR of internal elements, Southern blotting,comparative genomic hybridization (CGH), sequencing and etc. In theexample, correct lox2272-lox2272 and loxP-loxP recombination alsoresults in two intact sets of piggyBac elements that did not exist priorto insertion. An intact piggyBac element is comprised of a set ofinverted repeats which are depicted in the figure by “PB5′” and “PB3′”.An appropriated oriented set of piggyBac elements are the substrate ofpiggyBac transposase which can catalyse recombination between theelements, resulting in deletion of intervening sequences as well as bothelements. The DNA remaining after a piggyBac transposition is leftintact and is lacking any remnant of the piggyBac element. In theexample, ES cell clones with successful piggyBac transposition areselected by loss of the active puroTK element which renders the cellsresistant to the drug FIAU (FIGS. 4c and 4d ).

The final product of the SRMCE method in this example is a IGH locuswith several polymorphic V gene segments inserted along with a set ofendogenous unmodified VH gene segments between sequences of the mousegenome on the 5′ side and the mouse IGH constant region gene segments onthe 3′ side. The polymorphic V gene segments are positioned such thatthey can participate in the recombination events associated with B cellmaturation yielding VDJ gene segments. These gene segments can then betranscribed and spliced to the mouse constant region. Translation ofthese transcripts will result in the production of an antibody heavychain encoded by the polymorphic V gene segment, a human DH genesegment, a human JH gene segment and a mouse constant heavy chain genesegment.

As is well known to those skilled in the art, an ES cell clone can beused to create a line of genetically modified mice via injection of saidcells into a mouse blastocyst embryo, transferring the injected embryoto a suitable recipient and breeding the chimeric offspring that result.The modified gene locus can be propagated through breeding and madeeither heterozygous or homozygous depending on the genetic cross.

It is evident from the structure of the IGH locus provided in thisexample and by knowledge of the mechanisms involved in B cell receptor(BCR) and antibody gene rearrangements that a large set of differentcombinations of polymorphic V gene segments with various DH and JH genesegments will result and these can contribute to a large repertoire offunctional antibody genes in a population of B cells in geneticallymodified animals. In this example, several different human VH1-69polymorphs are incorporated to provide superhuman VH diversity. Thisparticular VH gene segment is known to be prevalent in antibodies thatbind infectious disease pathogens (such as influenza virus) andtherefore the antibody repertoire of a mouse with the geneticmodification of this example would be expected to produce antibodieswith a bias in favour of those that bind infectious disease pathogens.The repertoire, in other words, would have a larger subset of antibodieswith superior affinities for pathogen antigens. Examples of suchpathogens include influenza virus, hepatitis C virus (HCV) and humanimmunodeficiency virus-1 (HIV-1) (see also table above).

Example 3 Alignment of 13 VH1-69 Alleles

Building a more diverse antibody repertoire by incorporating additionalV gene segment polymorphs requires availability of polymorphic variantsof V gene segments. One source of such variants include sequencedatabases. In this example, 13 distinct variants of the VH1-69 genesegment are provided.

These variant sequences and comparisons are drawn from the“IMmunoGeneTics” IMGT Information System (www.imgt.com) database. FIG. 5is a diagram of the alignment of variants *02 through *13 with the *01variant. The VH1-69*01 nucleotide and amino acid sequence is provided atthe top of the figure. Where the remaining variants are identical to the*01 variant sequence a dash is inserted below the sequence. Nucleotidedifferences are noted alongside the appropriate variant and if thesequence change results in a protein coding change, the amino acidchange is indicated above the triplet.

FIG. 5 depicts between 1 and 4 amino acid changes for each variant incomparison to the *01 variant. All of the amino acid changes occur inthe part of the heavy chain protein encoding the complementaritydetermining regions (CDRs). These regions are responsible for antigenspecificity and the affinity of the antibody for the antigen. It isevident that providing additional polymorphic CDRs in a repertoire ofantibodies will increase the likelihood of there being an antibody withsuperior binding characteristics for various antigens. In severalreports, it has been observed that the VH1-69-encoded variable region ofthe heavy chain is often found in antibodies that bind influenza virus,HCV and HIV-1 antigens (see table above). Therefore incorporating thepolymorphic V gene segments of this example into a transgenic animalmodel using the methods of Examples 1 and 2 would likely result in anantibody repertoire in said transgenic animal with more antibodies thatbind to antigens associated with these and other pathogens. And as isknown in the art, a larger repertoire increases the probability offinding monoclonal antibodies using, for example, hybridoma technology,that bind with high affinity and specificity to a desired antigen.

This disclosure therefore describes in these examples a transgenic mousemodel which can be immunized with pathogen or other antigens. Plasma Bcells from such an immunized mouse can be used to make a hybridomalibrary that can be screened for production of antibodies that bind thepathogen antigens. This library will be superior to libraries fromtraditional transgenic mice for finding such antibodies given theaddition of polymorphic VH1-69 gene segments to the IGH locus in saidtransgenic mouse.

These examples are not limiting to the human polymorphic V gene segmentsthat can be chosen or to the methods used to introduce them into ananimal model. The method can be used to construct a transgenic locuswith immunoglobulin D and/or J segments. The V, D, J segments can befrom a plurality of human sources (optionally more than one human ethnicpopulation).

Example 4 Human IgH JH Gene Variants Selected from the 1000 GenomesDatabase

Data is presented for human JH2, 5 and 6 variants. In Tables 10A, 11Aand 12A samples from humans from various populations are listed wherethe sequence analysis of the inventors has revealed the presence ofpolymorphisms in one or both IgH JH alleles. The population codes areexplained in Table 8 above. The polymorphisms are nucleotide variantsfrom JH2, 5 and 6 reference sequences (SEQ ID NOs: 1, 2 and 3respectively; see below). All references are sequences taken from theEnsembl database (www.ensembl.org). The JH5 reference is human IgHJ5-001 disclosed in that database. The JH6 reference is human IgH J6-001disclosed in that database. The JH2 reference is human IgH J2-001disclosed in that database.

The reference nucleotide and encoded amino acid sequences are shown onthe next page. Alignments with encoded amino acid sequences are alsoprovided, including the corresponding position numbers on humanchromosome 14.

Variant Frequencies are shown in Tables 10A, 11A and 12A and theserelate to the frequency of the variants in the 1000 Genomes Database(release current at October 2011).

Tables 10B, 11B and 12B show the non-synonymous nucleotide polymorphismsin the human JH variants, as sorted by the present inventors from the1000 Genomes database. Position numbers corresponding to nucleotidepositions on human chromosome 14 are shown for variant positions(chromosome 14 being the chromosome bearing the IgH locus in humans).Thus, for example, the first entry in Table 11B is “14:106330027:A/C”which refers to a position in a variant JH5 sequence wherein theposition corresponds to position 106,330,027 on human chromosome 14,such position being A (adenine) in the reference sequence. The “C”indicates that the present inventors observed a mutation to cytosine atthis position in the variants found in the 1000 Genomes database. Thischange leads to a change at the amino acid level of the encoded sequence(i.e., a “non-synonymous” change), in this case a change from a serine(found in the reference) to an alanine in the variant.

Example 5

Human Antibody Gene Segment Variant Identification & Population Analysis

The genomic coding region coordinates for each target gene for variantanalysis were identified from the Ensembl WWW site (www.ensembl.org)using coordinates from the GRCh.p8 Human Genome assembly(www.ncbi.nlm.nih.gov/projects/genome/assembly/grc). Using the collectedgene location coordinates, variant data was extracted from the publicftp site of the 1000 Genomes Project using the Perl Variant PatternFinder(VPF—www.1000genomes.org/variation-pattern-finder-api-documentation).

Data extracted by VPF was post processed using software to extract allnon-synonymous (NSS) variants with their associated genotype calls.Genotypes calls were assembled to form unique haplotypes, representinggroups of NSS variants associated with 1000 Genome population groups andfrequency of occurrence within those populations.

The output of the analysis results in tables such as in Table 13. Themain body of the table describes each haplotype in turn giving a uniqueID for that gene (in the range a-z,aa-zz), the population frequenciesand occurrence in individuals and unique population groups; one or moresubsequent columns describe the DNA base calls at each location thatform the haplotype giving both the base from the reference sequence orthe variant base call.

Table 13 was constructed in this manner. The table can be read asfollows:

The first four columns (left to right) consist of (1) the haplotype IDletter (ref indicates reference—the DNA base call at each genomiclocation from the GRCh37 Human Reference Assembly) (2) the observedcumulative frequency of the haplotype among the different populations(3) the number of individuals in which a specific haplotype was observed(4) the number of unique population groups that the identifiedindividuals belong to (the actual population group identifiers aredisplayed as a string of ID's in the most right hand column for eachhaplotype. For example haplotype ‘a’ has a population ID string of ‘3,4, 9, 13’).

The populations are numbered as follows (population labels beingaccording to 1000 Genomes Project nomenclature)

1=ASW;

2=CEU;

3=CHB;

4=CHS;

5=CLM;

6=FIN;

7=GBR;

8=IBS;

9=JPT;

10=LWK;

11=MXL;

12=PUR;

13=TSI;

14=YRI.

Subsequent columns detail a single point variant and have the followingformat (top to bottom) (1) the human genomic location of the variant(format [chromosome number]:[location] e.g. ‘14:106204113’); (2) Theidentifier for the point variant as defined in DbSNP(www.ncbi.nlm.nih.gov/projects/SNP/); (3) One or additional rows showthe amino acid change as result of the variant for a specific transcript(denoted by the Ensembl transcript ID in the most right-hand column foreach row), the format is the amino acid in the reference sequencefollowed by ‘->’ and the amino acid caused by the substitution of thevariant in the reference sequence (e.g. ‘Gly->Arg’ means a that thetranslated reference sequence would result in a glycine at thatlocation, whereas the substitution of the identified variant wouldresult in translated protein containing arginine) using the IUPAC threeletter amino acid codes(http://paclupac.org/publications/pac/pdf/1972/pdf/3104x0639.pdf).Subsequent rows (one per haplotype) show the DNA base at each location,bases matching the reference sequence are shown in black on white background, bases varying from the reference are shown as white text on ablack background.

The most right-hand column contains the Ensembl transcript ID's (e.g.‘ENST00000390542’) for each of the gene transcript and relates to theamino acid changes to the left of this column. Because the transcriptsare differing lengths each variant position may or may not have anassociated amino acid change at the that position.

Example 6 Transgenic Mice, B-Cells, Hybridomas, Antibodies & HeavyChains Based on Human JH6*02

A functional human gene segment repertoire (from VH2-26 to JH6, see theIMGT database for the structure of the human IgH locus;

http://www.imgt.org/IMGTrepertoire/index.php?section=LocusGenes&repertoire=locus&species=human&group=IGK) was sectored by the inventors to produce two differenttransgenic heavy chain alleles (denoted S2F and S3F) and correspondingmice. The transgenic alleles were expressed in the mice and the heavychain repertoires were assessed at the RNA transcript level. Deepsequence analysis was carried out using Bioinformatics methods to assessV, D and JH gene usage, including in variable domain sequences having aHCDR3 length of at least 20 amino acids. Endogenous, mouse variableregion gene segments were inactivated by inversion (as per the methoddescribed in WO2011004192, this disclosure being incorporated herein byreference).

Sequencing of Human Donor DNA Samples: Identification of ConservedJH6*02 Variant

DNA samples from 9 anonymised consenting human donors were obtained bytaking cheek swabs.

The samples were processed and the DNA Samples were extracted follow theprotocol of QIAamp DNA Mini Kit (Cat. No. 51304, Qiagen).

PCR reactions were set up to amplify the JH6 region and PCR productswere sequenced (PCR Oligos sequence: Fwd. 5′-AGGCCAGCAGAGGGTTCCATG-3′(SEQ ID NO: 444), Rev. 5′-GGCTCCCAGATCCTCAAGGCAC-3′ (SEQ ID NO: 445)).

Sequence analysis was carried out by comparing to the JH6 referencesequence from IMGT annotated database (http://www.imgt.org/), and thisidentified that all 9 donor genomes contained the human JH6*02 variant,with this variant being in the homozygous state in 7 out of the 9donors. The inventors also consulted the genomic sequences publiclyavailable for Jim Watson and Craig Venter at Ensembl human genomedatabase [http://www.ensembl.org/]. These too contained the human JH6*02variant. This confirmed to the inventors that human JH6*02 is a common,conserved variant in humans, and thus a good candidate for constructionof a transgenic IgH locus as per the invention

Identification of Suitable Human DNA Sequence BACs

A series of human bacterial artificial chromosome (BAC) clones wereidentified from Ensemble (http://www.ensembl.org/index.html) or UCSC(http://genome.ucsc.edu/) human database searches based on gene name(IGH) or location (chromosome 14: 106026574-107346185). Seven human RP11BAC clones (see an extract of the UCSC database in FIG. 10, identifiedBACs being circled) were selected, RP11-1065N8 BAC carrying humanJH6*02. In total, the following BACs were identified as sources of humanIgH locus DNA: RP11-1065N8, RP11-659B19, RP11-14117, RP-112H5,RP11-101G24, RP11-12F16 and RP11-47P23.

With a similar approach, different BAC clones (eg, different RP11 cloneIDs or different sources from RP11) or genetically engineered BACs canbe selected for insertion into the mouse IGH locus to provide differentsets of human repertoires in the transgenic mouse.

Construction of Transgenic IgH Loci

Insertion of human heavy gene segments from a 1st IGH BAC (RP11-1065N8)into the IGH locus of mouse AB2.1 ES cells (Baylor College of Medicine)was performed to create a heavy chain allele denoted the 51 allele. Theinserted human sequence corresponds to the sequence of human chromosome14 from position 106494908 to position 106328951 and comprisesfunctional heavy gene segments VH2-5, VH7-4-1, VH4-4, VH1-3, VH1-2,VH6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15,D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25,D1-26, D7-27, JH1, JH2, JH3, JH4, JH5 and JH6 (in 5′ to 3′ order),wherein the JH6 was chosen to be the human JH6*02 variant. The insertionwas made between positions 114666435 and 114666436 on mouse chromosome12, which is upstream of the mouse Ĉ region. The mouse VH, D and J Hgene segments were retained in the locus, immediately upstream of (5′of) the inserted human heavy chain DNA.

A second allele, S2 was constructed in which more human functional VHgene segments were inserted upstream (5′) of the 5′-most VH inserted inthe S1 allele by the sequential insertion of human DNA from a second BAC(BAC2). The inserted human sequence from BAC2 corresponds to thesequence of human chromosome 14 from position 106601551 to position106494909 and comprises functional heavy chain gene segments VH3-13,VH3-11, VH3-9, VH1-8, VH3-7. The mouse VH, D and JH gene segments wereretained in the locus, immediately upstream of (5′ of) the insertedhuman heavy chain DNA. In a subsequent step, these were inverted toinactivate them, thereby producing S2F mice in which only the humanheavy chain variable region gene segments are active.

A third allele, S3 was constructed in which more human functional VHgene segments were inserted upstream (5′) of the 5′-most VH inserted inthe S2 allele by the sequential insertion of human DNA from a third BAC(BAC3). The inserted sequence corresponds to the sequence of humanchromosome 14 from position 106759988 to position 106609301, andcomprises functional heavy chain gene segments, VH2-26, VH1-24, VH3-23,VH3-21, VH3-20, VH1-18, and VH3-15. The mouse VH, D and JH gene segmentswere retained in the locus, immediately upstream of (5′ of) the insertedhuman heavy chain DNA. In a subsequent step, these were inverted toinactivate them, thereby producing S3F mice in which only the humanheavy chain variable region gene segments are active.

Mice bearing either the S2F or S3F insertion into an endogenous heavychain locus were generated from the ES cells using standard procedures.The other endogenous heavy chain locus was inactivated in the mice byinsertion of an inactivating sequence comprising neoR into the mouseJH-Ĉ intron (to produce the “HA” allele).

Immunisation Procedure

Transgenic mice of the S2F or S3F genotype were primed with 20-40 ugrecombinant proteins obtained commercially or produced in house withAntigen 1 (OVA (Sigma A7641); Antigen 2 (a human infectious diseasepathogen antigen) and Antigen 3 (a human antigen) via the ip route incomplete Freunds adjuvant (Sigma F 5881) and 10 ug/animal CpG (CpGoligo; Invivogen, San Diego, Calif., USA) and then boosted twice inabout two weekly intervals with about half the amount of antigen inincomplete Freunds adjuvant (Sigma F 5506) and 10 ug/animal CpG. Finalboosts were administered two weeks later iv without any adjuvant andcontained 5-10 ug protein in PBS.

Hybridoma Fusion Procedure

Spleens were taken 3 days after the final boost and splenocytes weretreated with CpG (25 ̂m final concentration) for and left until thefollowing day. Cells were then fused with SP0/2 Ag14 myeloma cells (HPACultures Cat No 85072401) using a BTX ECM2001 electrofusion instrument.Fused cells were left to recover for 20 minutes then seeded in a T75flask until next morning. Then the cells were spun down and plated outby dilution series on 96-well culture plates and left for about 10 daysbefore screening. Media was changed 1-3 times during this period.

Screening

Culture supernatants of the hybridoma wells above were screened usinghomogeneous time resolved fluorescence assay (htrf) using Europiumcryptate labelled anti-mouse IgG (Cisbio anti-mouse Ig EuropiumCryptate) and a biotin tagged target antigen with a commerciallyavailable streptavidin conjugated donor (Cisbio; streptavidin conjugatedD2) or by IgG-specific 384 well ELISA. Positive wells identified by htrfwere scaled to 24-well plates or immediately counterscreened using anIgG-specific detection ELISA method. Positives identified by primaryELISA screen were immediately expanded to 24-well plates. Once cultureswere expanded to 24-well stage and reached confluency, supernatants werere-tested using htrf or IgG-specific ELISA to confirm binding to targetantigen. Supernatant of such confirmed cultures were then also analysedby surface plasmon resonance using a BioRad ProteOn XPR36 instrument.For this, antibody expressed in the hybridoma cultures was captured on abiosensor GLM chip (BioRad 176-512) which had an anti-mouse IgG (GEHealthcare BR-1008-38)) covalently coupled the biosensor chip surface.The antigen was then used as the analyte and passed over the capturedhybridoma antibody surface. For Antigen 2 and Antigen 3, concentrationsof 256 nM, 64 nM, 16 nM, 4 nM and 1 nM were typically used, for Antigen1, concentrations of 1028 nM, 256 nM, 64 nM, 16 nM and 4 nM weretypically used, binding curves were double referenced using a 0 nMinjection (i.e. buffer alone). Kinetics and overall affinities weredetermined using the 1:1 model inherent to the BioRad ProteOn XPR36analysis software.

Any clones with confirmed binding activity were used for preparing totalRNA and followed by PCR to recover the heavy chain variable regionsequences. Standard 5′-RACE was carried out to analyse RNA transcriptsfrom the transgenic heavy chain loci in the S2F and S3F mice.Additionally, deep sequence analysis of almost 2000 sequences producedby the mice was carried out.

Bionformatics Analysis

Sequences for analysis were obtained from two different methods:

The first is from RNA extracted from the spleen: first cDNA strand wassynthesized using an oligo based on the Cmu region of the mouse IGHlocus as a PCR template. PCR was performed using this oligo with anoligo dT-anchor primer. Then PCR product was cloned into pDrive vector(Qiagen) and then sequenced.

The second is from hybridomas generated through electro-fusion: totalRNA was extracted from hybridoma lines of interest using standard Trizolmethods and frozen at −80° C. for long term storage. cDNA was generatedfrom 100 ng total RNA using standard Superscript III reversetranscriptase and a gene-specific reverse primer binding to all mouseIgG isotypes for heavy chain and a mouse kappa constant region primerfor the light chain amplification. 2-3 ul of cDNA were then used astemplate in a PCR reaction using Pfu DNA polymerase and a panel ofdegenerate forward primers annealing to the leader sequence of the humanimmunoglobulin variable domain as well as one mouse pan-IgG reverseprimer. PCR products were run out of a 1% agarose gel and bands ofapproximately 350-450 basepairs extracted and purified. DNA was thensequenced.

The sequences from the first method can either be from IgM from Naivemice or IgG from immunised mice. The samples from the second method areall from IgG from immunised mice, and specific to the immunisingantigen. Almost 2000 sequences were analysed.

The sequences were obtained as a pair of forward and reverse reads.These were first trimmed to remove low-quality base calls from the endsof the reads (trimmed from both ends until a 19 nucleotide window had anaverage quality score of 25 or more). The reads were combined togetherby taking the reverse complement of the reverse read, and aligning itagainst the forward read. The alignment scoring was 5 for a match, −4for a mismatch, a gap open penalty of 10 and a gap extension penaltyof 1. A consensus sequence was then produced by stepping through thealignment and comparing bases. When there was a disagreement the basewith the highest quality value from sequencing was used.

The BLAST© (Basic Local Alignment Search Tool) (Camacho C., CoulourisG., Avagyan V., Ma N., Papadopoulos J., Bealer K., & Madden T. L. (2008)“BLAST©: architecture and applications.” BMC Bioinformatics 10:421http://www.ncbi.nlm.nih.gov/pubmed/20003500) program ‘blastn’ was thenused to find the germline J and V segments used in each sequence. Awordsize of 30 was used for V matching, and 15 for J matching. Thedatabase searched against was constructed from the NGS sequencing of theBACs which were used to generate the Kymouse.

If a sequence matched both a V and a J segment, the sequence between thetwo was then compared to a database of germline D segments in the mouseusing ‘blastn’ with a wordsize of 4 and the options ‘blastn-short’ and‘ungapped’. This was used to assign a D segment, if possible. The CDR3was identified by searching for the conserved “TATTACTGT” sequence inthe V segment, and the “CTGGGG” in the J segment. If these motifs werenot found, then up to 4 mismatches were allowed. The IMGT definition ofCDR3 was used, so the CDR3 length is calculated from after the “TGT” inthe V to before the “TGG” in the J. Sequences with an out of framejunction (those which do not have a CDR3 nucleotide length divisible by3) or which contained a stop codon (“TAA”, “TAG” or “TGA”) wereexcluded.

The identity of the matching V, J and D segments as well as the CDR3length from this assignment were then saved as a table for downstreamanalysis. The ratio of IGHJ6*02 used increased from the naive toimmunised mice, as well as being enriched in the sub-population ofsequences with a long HCDR3 (defined as consisting of 20 or more aminoacids):

All HCDR3 >20 Total Total JH6*02% Count JH6*02% Count % HCDR3 >20 Naive22.31% 1340 91.11% 45 3.36% Immunised 37.50% 256 66.67% 9 3.52%Hybridoma 36.13% 119 63.64% 11 9.24%

This shows that the JH6*02 gene segment is selected for by immunisation,as the proportion of JH6*02 usage increases after immunisation. JH6*02is also used in the majority of antibodies with a long HCDR3 length,which is desirable for targets which are specifically bound by longHCDR3 length antibodies.

SEQ ID NO: 1 (JH5 Reference) T T G A C C A A G C T G G G G A C C C C G GT C C C T T G G G A C C A G T G G C A G A G G A G T C JH5 Alignment:(top line = SEQ ID NO: 1, Middle line = SEQ ID NO: 5, Bottom line =SEQ ID NO: 6) 106,330,068 LgH J5001 106,330,072 106,330,071 106,330,067106,330,041 106,330,082 106,330,027 106,330,024 106,330,065 106,330,065106,330,063 106,330,062 106,330,045 106,330,044 T T G A C C A A G C T GG G G A C C C C G C C T T G G G A C C A G T G G C A G A G G- A A C T G GT T C G A C C C C T G G C T G G - C A C C G T C C T C A G W P W G Q G TL V T V S S SEQ ID NO: 2 (JH6Reference) A T G A T G A T G A T G A T G AT G T A C C T G C A G A C C C C G T T T C C C T G G T G C C A G T G G CA G A G G A G T JH6 Alignment: (top line = SEQ ID NO: 2, Middle line =SEQ ID NO: 7, Bottom line = SEQ ID NO: 8) LgH J6001 106,329,468106,329,453 106,329,452 106,329,451 106,329,435  T T G A C C A A G C T GG G G A C C C C G C C T T G G G A C C A G T G G C A G A G G- A A106,329,426 106,329,419 106,329,417 106,329,414 106,329,413 106,329,411106,329,408 C C C T G G T G C C A G T G G C A G A G G A G T C G G G A CC A C G G T C A C C G T C T C C T C A G G T T V T V S SSEQ ID NO: 3 JH 2 Reference) A T G A C C A T G A A G C T A G A G A C C CC G G C A C C G T G G G A C C A G T G A C A G A G G A G T CJH2 Alignment: (top line = SEQ ID NO: 3, Middle lien =SEQ ID NO: 9, Bottom line = SEQ ID NO: 10) IgH J5-001 106,331,460106,331,455 106,331,453 106,331,453 A T G A C C A T G A A G C T A G A GA C C C C G G C A C C G T G G G A C C A G T G A C A G A G G A G T C T AC T G G A C T T C G A C T C T G G G G C C G T G G C A C C C T G G T C AC T G T C T C C T C A G Y W Y F D L W G R G T L V T V S S

Tables

In the tables, the notation is illustrated by the following example

IGLV1 40 G1 40*02 X53936 :g9>|c1)>g,L4>VI

Polymorphic variant IGV lambda VI-40*02 has Genbank Accession No. X53936and when compared to the *01 variant, the VI-40*02 variant has mutationsat positions 9, 10 and 4. For example, at position 9, a “C” appearsinstead of a “G” that is present in the *01 variant. The “I” is simply anotation separator, and does not indicate any mutation. For example the“g282 I” notation indicates no change (ie, position 282 is a g). “del#”means that the residue at that position is absent.

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REFERENCES

-   1. Nat Biotechnol. 2005 September; 23(9):1117-25; Human antibodies    from transgenic animals; Lonberg N.-   2. J Clin Invest. 1992 March; 89(3):729-38; Immunoglobulin light    chain variable region gene sequences for human antibodies to    Haemophilus influenzae type b capsular polysaccharide are dominated    by a limited number of V kappa and V lambda segments and VJ    combinations; Adderson E E, Shackelford P G, Insel R A, Quinn A,    Wilson P M, Carroll W L.-   3. J Immunol. 1993 October 15; 151(8):4352-61; Clonal    characterization of the human IgG antibody repertoire to Haemophilus    influenzae type b polysaccharide. V. In vivo expression of    individual antibody clones is dependent on Ig CH haplotypes and the    categories of antigen; Chung G H, Scott M G, Kim K H, Kearney J,    Siber G R, Ambrosino D M, Nahm M H.-   4. J Immunol. 1998 December 1; 161(11):6068-73; Decreased frequency    of rearrangement due to the synergistic effect of nucleotide changes    in the heptamer and nonamer of the recombination signal sequence of    the V kappa gene A2b, which is associated with increased    susceptibility of Navajos to Haemophilus influenzae type b disease;    Nadel B, Tang A, Lugo G, Love V, Escuro G, Feeney A J.-   5. J Clin Invest. 1996 May 15; 97(10):2277-82; A defective Vkappa A2    allele in Navajos which may play a role in increased susceptibility    to haemophilus influenzae type b disease; Feeney A J, Atkinson M J,    Cowan M J, Escuro G, Lugo G.-   6. Infect Immun. 1994 September; 62(9):3873-80; Variable region    sequences of a protective human monoclonal antibody specific for the    Haemophilus influenzae type b capsular polysaccharide; Lucas A H,    Larrick J W, Reason D C.-   7. J Clin Invest. 1993 June; 91(6):2734-43; Restricted    immunoglobulin VH usage and VDJ combinations in the human response    to Haemophilus influenzae type b capsular polysaccharide. Nucleotide    sequences of monospecific anti-Haemophilus antibodies and    polyspecific antibodies cross-reacting with self antigens; Adderson    E E, Shackelford P G, Quinn A, Wilson P M, Cunningham M W, Insel R    A, Carroll W L.-   8. J Clin Invest. 1993 March; 91(3):788-96; Variable region    expression in the antibody responses of infants vaccinated with    Haemophilus influenzae type b polysaccharide-protein conjugates.    Description of a new lambda light chain-associated idiotype and the    relation between idiotype expression, avidity, and vaccine    formulation. The Collaborative Vaccine Study Group; Granoff D M,    Shackelford P G, Holmes S J, Lucas A H.-   9. Infect Immun. 1994 May; 62(5):1776-86; Variable region sequences    and idiotypic expression of a protective human immunoglobulin M    antibody to capsular polysaccharides of Neisseria meningitidis group    B and Escherichia coli K1; Azmi F H, Lucas A H, Raff H V, Granoff D    M.-   10. J Clin Invest. 1992 December; 90(6):2197-208; Sequence analyses    of three immunoglobulin G anti-virus antibodies reveal their    utilization of autoantibody-related immunoglobulin Vh genes, but not    V lambda genes; Huang D F, Olee T, Masuho Y, Matsumoto Y, Carson D    A, Chen P P.-   11. Science. 2011 August 12; 333(6044):834-5, Biochemistry. Catching    a moving target, Wang T T, Palese P-   12. Science. 2009 Apr. 10; 324(5924):246-51. Epub 2009 February 26;    Antibody recognition of a highly conserved influenza virus epitope;    Ekiert D C, Bhabha G, Elsliger M A, Friesen R H, Jongeneelen M,    Throsby M, Goudsmit J, Wilson I A.-   13. PLoS One. 2008; 3(12):e3942. Epub 2008 Dec. 16; Heterosubtypic    neutralizing monoclonal antibodies cross-protective against H5N1 and    H1N1 recovered from human IgM© memory B cells; Throsby M, van den    Brink E, Jongeneelen M, Poon L L, Alard P, Cornelissen L, Bakker A,    Cox F, van Deventer E, Guan Y, Cinatl J, ter Meulen J, Lasters I,    Carsetti R, Peiris M, de KruifJ, Goudsmit J.-   14. Nat Struct Mol Biol. 2009 March; 16(3):265-73. Epub 2009 Feb.    22, Structural and functional bases for broad-spectrum    neutralization of avian and human influenza A viruses, Sui J, Hwang    W C, Perez S, Wei G, Aird D, Chen L M, Santelli E, Stec B, Cadwell    G, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox N J, Bankston L    A, Donis R O, Liddington R C, Marasco W A.-   15. Science. 2011 August 12; 333(6044):843-50. Epub 2011 Jul. 7, A    highly conserved neutralizing epitope on group 2 influenza A    viruses, Ekiert D C, Friesen R H, Bhabha G, Kwaks T, Jongeneelen M,    Yu W, Ophorst C, Cox F, Korse H J, Brandenburg B, Vogels R,    Brakenhoff J P, Kompier R, Koldijk M H, Cornelissen L A, Poon L L,    Peiris M, Koudstaal W, Wilson I A, Goudsmit J.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180030121A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A method for producing a heavy chain, VH domain or an antibodyspecific to a target antigen, wherein the heavy chain, VH domain orantibody comprises an HCDR3 at least 20 amino acids in length, themethod comprising providing a non-human vertebrate, optionally a mouseor a rat, that has a genome comprising an immunoglobulin heavy chainlocus comprising unrearranged human gene segment JH6*02, one or more VHgene segments and one or more D gene segments upstream of a constantregion; wherein the gene segments in the heavy chain locus are operablylinked to the constant region thereof so that the vertebrate is capableof producing an antibody heavy chain produced by recombination of thehuman JH6*02 with a human D segment and a human VH segment, wherein theJH6*02 gene segment comprises the following nucleotide sequence: ATTACTACTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG, andwherein the vertebrate has been immunised with the target antigen andproduces an antibody heavy chain specific for the target antigen whereinthe variable domain of the heavy chain is the product of recombinationbetween a human VH, D and JH6*02 and wherein the HCDR3 length is atleast 20 amino acids, and isolating from the non-human vertebrate theheavy chain, VH domain or an antibody specific to the target antigen ora cell producing the heavy chain, VH domain or antibody, wherein theheavy chain, VH domain or antibody comprises a HCDR3 that is derivedfrom the recombination of human JH6*02 with a human VH gene segment anda human D gene segment, optionally wherein the constant region of thelocus is a non-human vertebrate (e.g., mouse or rat) constant region,and the non-human constant region of the isolated heavy chain orantibody is replaced with a human constant region.
 2. The method ofclaim 1 comprising isolating a heavy chain, VH domain or an antibody,wherein the HCDR3 length is at least 20 amino acids.
 3. The method ofclaim 1, comprising isolating a B-cell or hybridoma expressing a heavychain VH domain that is identical to the VH domain of the heavy chain ofclaim
 2. 4. The method of claim 1, wherein the constant region is mouseor rat.
 5. The method of claim 1, comprising removing B lymphocytes fromthe vertebrate and selecting one or more B lymphocytes expressingantibodies that bind to the antigen, optionally immortalising saidselected B lymphocytes or progeny thereof, optionally by producinghybridomas therefrom, and isolating an antibody expressed by the Blymphocytes.
 6. The method of claim 5, further comprising isolating fromsaid B lymphocytes nucleic acid encoding said antibody.
 7. The method ofany claim 1, wherein the constant region of the locus is a non-humanvertebrate (e.g., mouse or rat) constant region, and the method furthercomprises replacing the non-human constant region of the isolated heavychain or antibody with a human constant region.
 8. The method of claim6, further comprising exchanging the heavy chain constant regionnucleotide sequence of the antibody with a nucleotide sequence encodinga human or humanised heavy chain constant region, and optionallyaffinity maturing the variable region of said antibody.
 9. The method ofclaim 8, further comprising inserting said nucleic acid into anexpression vector and optionally a host cell.
 10. The method of claim 9,comprising expressing the heavy chain, VH domain or antibody from thehost cell and providing an isolated heavy chain, VH domain or antibody.11. The method of claim 1, wherein the locus comprises one, more or allhuman D gene segments D3-9; D4-17; D3-10; D2-2; D5-24; D6-19; D3-22;D6-13; D5-12; D1-26; D1-20; D5-18; D3-16; D2-21; D1-14; D7-27; D1-1;D6-25; D2-14; and D4-23, optionally wherein the locus comprises one,more or all human D gene segments D3-9, D3-10, D6-19, D4-17, D6-13,D3-22, D2-2, D2-25 and D3-3.
 13. The method of claim 1, wherein thelocus comprises a plurality of human D gene segments and the JH6*02 isin human germline configuration with respect to the 3′-most human D genesegment.
 14. The method of claim 1, wherein the locus comprises one,more or all of human gene segments selected from V3-21, V3-13, V3-7,V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18, V4-4, V3-9, V3-23, V3-11, V3-20,D3-9*01, D3-10*01, D6-19*01, D6-13*01, D1-26*01, IGHV1-8*01,IGHV4-61*01, IGHV6-1*01, IGHV4-4*02, IGHV1-3*01, IGHV3-66*03, IGHV3-7*01and IGHV3-9*01.
 15. The method of claim 1, wherein the antibody heavychain is a product of the recombination of JH6*02 with a human VH genesegment recited in claim 14 and/or a D gene segment recited in claim 11.16. The method of claim 1, wherein all endogenous non-human vertebrateheavy chain variable region gene segments have been inactivated in thegenome and/or wherein the genome is homozygous for said heavy chainlocus.
 17. The method of claim 1, wherein the vertebrate is a mousecomprising functional heavy gene segments VH2-5, VH7-4-1, VH4-4, VHI-3,VHI-2, VH6-1, DI-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, DI-14,D2-15, D3-16, D4-17, D5-18, D6-19, DI-20, D2-21, D3-22, D4-23, D5-24,D6-25, DI-26, D7-27, JHI, JH2, JH3, JH4, JH5 and JH6, in 5′ to 3′ order,wherein the JH6 is the human JH6*02 variant, wherein the insertion ofhuman DNA is made between positions 114666435 and 114666436 on mousechromosome 12; wherein human functional heavy chain gene segmentsVH3-13, VH3-11, VH3-9, VH1-8, VH3-7 are inserted upstream of VH2-5;wherein the mouse VH, D and JH gene segments are retained in the locus,immediately upstream of (5′ of) the inserted human heavy chain DNA; andwherein the mouse VH, D and J segments are inverted to inactivate them,thereby producing mice in which only the human heavy chain variableregion gene segments are active.
 18. The method of claim 17 additionallycomprising VH2-26, V H 1-24, VH 3-23, VH 3-21, VH3-20, VH1-18, and VH3-15 inserted upstream (5′) of the 5′-most VH.
 19. The method of claim1, wherein (i) the target antigen is an antigen of an infectious diseasepathogen; (ii) the target antigen is a receptor, and the antibody heavychain specifically binds a receptor cleft; or (iii) the target antigenis an enzyme, and the antibody heavy chain specifically binds the enzymeactive site.
 20. A non-human vertebrate cell having a genome comprising:at least 3 human variable region gene segments of the same type, whereinat least two of the human gene segments are variants that are notidentical to each other; at least 2 different human variable region genesegments of the same type in cis at the same Ig locus; at least 2different human variable region gene segments of the same type in transat the same Ig locus, or first and second human Ig locus gene segmentsof the same type, wherein the first gene segment is a gene segmentselected from any one of Tables 1 to 7 and 9 to 14 and the second genesegment is the corresponding reference sequence.
 21. A method ofproviding an enhanced human immunoglobulin variable region gene segmentrepertoire, the method comprising providing a population of non-humanvertebrates comprising a repertoire of human variable region genesegments, providing at least 2 different human variable region genesegments of the same type, wherein a first of said different genesegments is provided in the genome of a first vertebrate of thepopulation, and a second of said different gene segments is provided inthe genome of a second vertebrate of the population, wherein the genomeof the first vertebrate does not comprise the second gene segment.
 22. Alibrary of antibody-producing transgenic cells whose genomescollectively encode a repertoire of antibodies, wherein (a) a firsttransgenic cell expresses a first antibody having a chain encoded by afirst immunoglobulin gene, the gene comprising a first variable domainnucleotide sequence produced following recombination of a first humanunrearranged immunoglobulin gene segment; (b) a second transgenic cellexpresses a second antibody having a chain encoded by a secondimmunoglobulin gene, the second gene comprising a second variable domainnucleotide sequence produced following recombination of a second humanunrearranged immunoglobulin gene segment, the first and secondantibodies being non-identical; (c) the first and second gene segmentsare different and derived from the genome sequences of first and secondhuman individuals respectively, wherein the individuals are different;wherein the second human immunoglobulin gene segment is a polymorphicvariant of the first human immunoglobulin gene segment and wherein thesecond gene segment is selected from the group consisting of a genesegment in any of Tables 1 to 7 and 9 to 14, and wherein (d) the cellsare non-human vertebrate cells.
 23. A non-human vertebrate cellcomprising a genome that comprises (i) a transgenic heavy chainimmunoglobulin locus, wherein the locus comprises a full humanrepertoire of functional VH, D and JH segments derived from the genomesequence of the same human individual, supplemented with one or moreadditional functional human VH, D and/or JH gene segment that is notfound in the genome sequence of said human individual; (ii) a transgenickappa light chain immunoglobulin locus, wherein the locus comprises afull human repertoire of functional Vk and Jk segments derived from thegenome sequence of the same human individual, supplemented with one ormore additional functional human Vk and/or Jk gene segment that is notfound in the genome sequence of said human individual; or (iii) atransgenic lambda light chain immunoglobulin locus, wherein the locuscomprises a full human repertoire of functional V lambda and J lambdasegments derived from the genome sequence of the same human individual,supplemented with one or more additional functional human V lambdaand/or J lambda gene segment that is not found in the genome sequence ofsaid human individual.
 24. A transgenic immunoglobulin locus comprisinga synthetic immunoglobulin gene haplotype, the haplotype comprisingfirst and second human gene segments (each being a V, D or J), a switchregion and a constant region, wherein a) the second gene segment is apolymorphic variant of the first gene segment; or b) the first andsecond gene segments are derived respectively from genome sequence ofindividuals from different, first and second, ethnic populationsaccording to the 1000 Genomes database and the second gene segment isnot found in the first population according to the 1000 Genomesdatabase; and wherein the constant region, and optionally the switchregion, are non-human vertebrate constant and switch regions.
 25. Amethod of producing an antibody heavy chain, the method comprisingproviding an antigen-specific heavy chain variable domain; and combiningthe variable domain with a human heavy chain constant region to producean antibody heavy chain comprising (in N- to C-terminal direction) thevariable domain and the constant region; wherein the human heavy chainconstant region is an IGHAref, IGHAIa, IGHA2a, IGHA2b, IGHGIref,IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref, IGHG4a, IGHDref,IGHEref, IGHMref, IGHMa or IGHMb constant region.
 26. A non-human cell(eg, a mouse cell or rat cell) comprising a genome that comprises atransgenic heavy chain immunoglobulin locus, wherein the locus comprisesat least 42 (optionally at least 43, 44, 45, 46, 47, 48, 49, 50)functional human VH gene segments, one or more functional human D genesegments, one or more functional human JH gene segments operablyconnected upstream of a non-human vertebrate constant region (eg, amouse constant region, eg, a Cmu and/or a C gamma).
 27. A non-human cell(eg, a mouse cell or rat cell) comprising a genome that comprises atransgenic kappa light chain immunoglobulin locus, wherein the locuscomprises at least 39 functional human Vk gene segments and one or morefunctional human Jk gene segments operably connected upstream of anon-human vertebrate constant region (eg, a mouse constant region). 28.A non-human cell (eg, a mouse cell or rat cell) comprising a genome thatcomprises a transgenic lambda light chain immunoglobulin locus, whereinthe locus comprises at least 32 functional human V lambda gene segmentsand one or more functional human J lambda gene segments operablyconnected upstream of a non-human vertebrate constant region (eg, amouse constant region).